A hair follicle detection kit, method and application thereof

Abstract

The present invention relates to the field of medical detection technology, and in particular to a hair follicle detection kit, method and application thereof. The hair follicle detection kit of the present invention comprises a hair follicle treatment solution and a dyeing solution, and the hair follicle treatment solution comprises Triton X-100 and a solvent, or comprises Triton X-100, type I collagenase and a solvent; wherein, the volume percentage concentration of Triton X-100 is 0.1-0.5%, and the mass percentage concentration of type I collagenase is 0.01-0.5%. The present invention combines Triton X-100 and type I collagenase in a specific ratio for treating in vitro hair follicles, increasing the permeability and transmittance of the hair follicles, and being more conducive to the penetration of the dyeing solution into the tissue. The detection method after treatment by the present invention not only significantly shortens the detection time, but also reduces the risk of damage to hair follicles or cells during the slicing process, and can provide complete information on the hair follicle tissue structure and hair follicle blood vessels. The method of the present invention only takes half an hour to complete the detection of hair follicles and issue a test report.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a hair follicle detection kit, method and application thereof. Background Art

[0002] Hair loss and baldness have become the biggest problems that plague modern people, and more and more young people have joined the ranks of hair loss. In the past few decades, the treatment of hair loss has only focused on the hair. In order to have healthy and thick hair, many people spend a lot of money on various high-end shampoo and hair care products. However, simply maintaining hair without dealing with the causes of hair loss cannot improve and treat hair loss at all.

[0003] Common factors that cause hair loss include genetic factors, disease, hormonal changes, chemotherapy drugs, stress, malnutrition, excessive hairdressing and aging. Although there are many reasons for hair loss, no matter what the cause is, it all comes down to a problem with the hair follicles.

[0004] The hair follicle is a biological factory for hair processing, which consists of the hair shaft, inner root sheath, companion layer, outer root sheath, hair matrix, bulge and dermal interstitial part (dermal sheath and dermal papilla). The function of hair follicles depends on more than 20 types of cells in the hair follicles. Among them, the five most critical types of cells are hair follicle stem cells and melanocyte stem cells located in the bulge, dermal papilla cells located in the dermal papilla, and hair matrix cells and melanocytes located in the hair matrix. Among them:

[0005] Hair follicle stem cells: Located in the bulge, they are the raw materials of most functional cells of the hair follicles, and continuously provide new cells for the growing hair follicles. Hair follicle stem cells are a type of adult stem cells, with the potential for self-replication and differentiation. The hair cycle is maintained by hair follicle stem cells. The hair follicle stem cells responsible for hair growth are usually in a dormant state, but they will quickly activate and divide in a new round of hair growth cycle. It is precisely because of the activation or dormant state of hair follicle stem cells that the normal shedding and regeneration of hair is guaranteed. If the hair follicle stem cells cannot be activated normally, or if the hair follicle stem cells are damaged or reduced in number, it will lead to hair loss.

[0006] Dermal papilla cells: The dermal microenvironment formed by dermal papilla cells has always been considered a key signal center for regulating hair follicle growth and rest. Dermal papilla cells are essential for activating hair follicle regeneration. During the growth induction period of hair follicles, dermal papilla cells can control the proliferation and differentiation of hair follicle stem cells by secreting regulatory signals. Prepare for the next hair cycle. Therefore, dermal papillae are necessary for hair growth. If dermal papillae are missing, hair follicles cannot be activated in the resting phase. In addition, the number of dermal papillae cells affects the size of hair follicles, and the size of dermal papillae is related to the thickness of hair.

[0007] Hair matrix cells: During the growth of hair, the hair matrix cells located in the hair matrix continue to divide and form the "raw materials" of hair. With the continuous supply of "raw materials", the hair shaft is pushed out of the scalp and becomes visible hair. The number of hair matrix progenitor cells and the size of the hair bulb will affect the density and thickness of hair.

[0008] Melanocyte stem cells and melanocytes: The state and quantity of these two cells play a decisive role in the color of hair. Melanocyte stem cells differentiate into melanocytes, and melanocytes synchronously produce melanin in the hair matrix, which is passed to the hair stem progenitor cells and their progeny, the medulla cells, the cortex cells, and the trichome cells, to color the hair and eventually form colored finished hair. If melanocyte stem cells cannot differentiate normally or the number of melanocytes and melanocyte stem cells decreases, it will cause hair to turn white.

[0009] Normal hair growth also requires microvessels around the hair follicles to supply nutrients to the hair follicles. The microvessels around the hair follicles are responsible for delivering a large amount of nutrients to the dermis where the hair follicles grow. The early growth period of the hair follicles is when their nutritional needs are the strongest, so the blood vessel density increases significantly. During the growth period, the blood vessels around the hair follicles are actively remodeled. If there are problems with the formation of blood vessels at this time, the supply of nutrients and oxygen for hair formation becomes insufficient. Fewer microvessels around the hair follicles or insufficient blood flow will lead to hair loss.

[0010] In summary, hair follicle circulation and hair regeneration require the cooperation and response of various cells in the hair follicles, the cells that synthesize hair need to remain active, cells need to be fully replenished after consumption, and blood vessels need to transport a large amount of nutrients to maintain hair follicle growth. Problems in any link will lead to hair follicle atrophy, and new hair will be thin, soft, yellow, and white; in severe cases, hair follicles will disappear completely, new hair cannot grow, and hair will become sparse and bald.

[0011] However, hair follicle detection is usually performed with a dermatoscope, which is actually a skin magnifying glass with a polarized light source. It can only detect the scalp and the hair that has grown out of the skin, but cannot detect whether there are structural defects in the hair follicles, whether the cells that make up the hair follicles are intact, and whether the blood supply around the hair follicles is sufficient.

[0012] Although the histopathological detection method (frozen section or paraffin section) can detect the structure and cells of hair follicles, it requires the hair follicles to be cut into 3-10μm slices and then stained, which only shows the local planar structure and cannot reflect the three-dimensional structure of hair follicle tissue and the reticular structure of hair follicle blood vessels. In addition, paraffin sections need to undergo tissue fixation, embedding, slicing and other processes, which takes a long time and the waiting time is long. Although frozen sections are quick to make, the freezing microtome is expensive and requires high slicing skills from the inspectors.

[0013] Therefore, there is an urgent need for a fast and efficient hair follicle detection kit and detection method to obtain complete information on the hair follicle tissue structure, hair follicle cells and microvessels around the hair follicles, so as to determine the integrity of the hair follicle tissue and cells and the blood supply of the hair follicles, and evaluate the growth status and regeneration ability of the hair follicles. Summary of the invention

[0014] In view of this, the present invention provides a hair follicle detection kit, method and application thereof. The hair follicle treatment solution in the hair follicle detection kit can effectively increase the permeability and light transmittance of the hair follicle, and is more conducive to dyeing and the detection of the hair follicle tissue structure, hair follicle cells and microvessels around the hair follicle after dyeing.

[0015] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0016] The invention provides a hair follicle detection kit, comprising a hair follicle treatment solution and a dyeing solution, wherein the hair follicle treatment solution comprises Triton X-100 and a solvent, or comprises Triton X-100, type I collagenase and a solvent; the dyeing solution is an NBT / BCIP dyeing solution or a combination of an NBT / BCIP dyeing solution and a hematoxylin dyeing solution; wherein the volume percentage concentration of Triton X-100 is 0.1-0.5%, and the mass percentage concentration of type I collagenase is 0.01-0.5%.

[0017] The hair follicles are treated with the hair follicle treatment solution in the kit of the present invention, which can form appropriate porosity and openings in the cell membrane, increase the permeability and light transmittance of the hair follicles, and facilitate the dye solution to penetrate into the tissue, so that it can be used for microscopic detection without sectioning.

[0018] In some embodiments, the hair follicle treatment solution comprises Triton X-100, type I collagenase and a solvent; the staining solution is NBT / BCIP staining solution or a combination of NBT / BCIP staining solution and hematoxylin staining solution; wherein the volume percentage concentration of Triton X-100 is 0.1 to 0.5%, and the mass percentage concentration of type I collagenase is 0.01 to 0.5%.

[0019] In some preferred embodiments, in the hair follicle treatment solution, the volume percentage concentration of Triton X-100 is 0.1-0.3%, specifically 0.1%, 0.2% or 0.3%; the mass percentage concentration of type I collagenase is 0.01-0.1%, specifically 0.01%, 0.05%, 0.1%.

[0020] When the mass percentage concentration of type I collagenase is too high (≥1%), it will destroy the hair follicle tissue structure, excessive dye will reside in the hair follicle tissue, the background color will become darker, the tissue transmittance will become worse, the contrast between the hair follicle structure and the blood vessels will be unclear, and the blood vessel distribution will be unclear, resulting in low image clarity and blood vessel staining effect scores. At the same time, it will also degrade the cell membrane and nuclear membrane, resulting in too deep staining, unclear contrast between the cell nucleus and cytoplasm, and inability to clearly observe the hair follicle cells. When the concentration of Triton X-100 is too low (≤0.05%), less dye enters the blood vessels, the blood vessel staining is incomplete, the lumen of the hair follicle blood vessels is unclear, and the image clarity and blood vessel staining effect scores after staining are low.

[0021] In some specific embodiments, the volume percentage concentration of Triton X-100 is 0.3%, and the mass percentage concentration of type I collagenase is 0.1%.

[0022] In some specific embodiments, the volume percentage concentration of Triton X-100 is 0.1%, and the mass percentage concentration of type I collagenase is 0.1%.

[0023] In some embodiments, the solvent is PBS buffer or double distilled water.

[0024] In some embodiments, the staining solution is NBT / BCIP staining solution.

[0025] In some embodiments, the staining solution is a combination of NBT / BCIP staining solution and hematoxylin staining solution. In this embodiment, the hair follicle detection kit of the present invention further includes a differentiation solution and a blueing solution.

[0026] In the present invention, in the NBT / BCIP solution, the concentration of NBT is 0.1-0.6 mg / ml, the concentration of BCIP is 0.05-0.3 mg / ml, and the solvent is PBS buffer.

[0027] In the present invention, the volume percentage of the hematoxylin stock solution in the hematoxylin solution is 50-100%.

[0028] The hair follicle detection kit of the present invention is used for detection without sectioning. After the hair follicles are cleaned, they are treated with a hair follicle treatment solution and then dyed, or the hair follicle treatment solution and the dyeing solution are used simultaneously.

[0029] The present invention provides a method for detecting in vitro hair follicles, which is to first treat the hair follicles with a hair follicle treatment solution and then dye them, and specifically comprises the following steps:

[0030] a1. Wash the hair follicles to be tested, treat them with hair follicle treatment solution for 8 to 20 minutes, wash them, and then stain them with NBT / BCIP staining solution for 3 to 15 minutes;

[0031] b1. Wash the stained hair follicles and place them on a test slide. Add 80-95% glycerol, seal the slide, and observe under a microscope.

[0032] In some embodiments, in step a1, the hair follicle treatment solution is used for a treatment time of 8 to 15 minutes.

[0033] In some specific embodiments, in step a1, the hair follicle treatment liquid treatment time is 10 minutes.

[0034] In some specific embodiments, the NBT / BCIP solution is used for staining for 10 minutes.

[0035] or,

[0036] a2. After washing the hair follicles to be tested, treat them with hair follicle treatment solution for 8 to 20 minutes, wash them, and then stain them with NBT / BCIP solution for 3 to 15 minutes, and then stain them with hematoxylin solution for 2 to 10 minutes;

[0037] b2. The stained hair follicles were treated with differentiation solution and blueing solution in turn, placed on a test slide, and 80-95% glycerol was added. The slide was sealed and observed under a microscope.

[0038] In some embodiments, in step a2, the hair follicle treatment solution treatment time is preferably 8 to 15 minutes, and in some specific embodiments, specifically 10 minutes.

[0039] In some specific embodiments, the NBT / BCIP solution staining time is 10 minutes.

[0040] In some specific embodiments, the hematoxylin solution staining time is 3 minutes.

[0041] The differentiation solution used in the differentiation is hydrochloric acid alcohol, wherein the volume percentage concentration of concentrated hydrochloric acid is 0.2-1.5%; the differentiation time is 0.5-5 minutes; wherein, the preparation method of the differentiation solution is: 0.2ml, 0.5ml, 1ml or 1.5ml of concentrated hydrochloric acid (37%) is dissolved in 200ml of 75% ethanol solution respectively, and stirred evenly to obtain a differentiation solution with a volume percentage concentration of concentrated hydrochloric acid of 0.2%, 0.5%, 1% or 1.5%.

[0042] The blueing liquid is an ammonia solution, and the mass percentage concentration of ammonia is 0.1-0.5%; the blueing time is 0.5-3 minutes. The preparation method of the ammonia blueing liquid of the present invention is as follows: 0.5 ml, 1 ml or 2.2 ml of 28% ammonia water is dissolved in 400 ml of double distilled water, and stirred evenly to obtain a blueing liquid with a mass percentage concentration of ammonia of 0.11%, 0.22% or 0.5%.

[0043] The present invention also provides a method for detecting in vitro hair follicles, which comprises the following steps when the hair follicle treatment solution and the dyeing solution are used simultaneously:

[0044] a3. After washing the hair follicles to be tested, add hair follicle treatment solution and NBT / BCIP staining solution and treat for 8 to 20 minutes;

[0045] b3. Wash the stained hair follicles and place them on a test slide. Add 80-95% glycerol, seal the slide, and observe under a microscope.

[0046] or,

[0047] a4. After washing the hair follicles to be tested, add hair follicle treatment solution and NBT / BCIP staining solution and treat for 8 to 20 minutes;

[0048] b4, stain the hair follicles after NBT / BCIP staining with hematoxylin solution for 2 to 10 minutes;

[0049] c4. The stained hair follicles were treated with differentiation solution and blueing solution in turn, placed on a test slide, and 80-95% glycerol was added. The slide was sealed and observed under a microscope.

[0050] After the hair follicles are processed and dyed using the detection method of the present invention, the length, diameter and melanin content of the hair follicles can be measured under a microscope, and the number of blood vessels around the hair follicles can be counted. It only takes half an hour to complete the detection of hair follicles and issue a test report. It is suitable for rapid detection before hair follicle transplantation, greatly shortening the traditional detection time, saving manpower and time costs and expensive antibody reagent costs. At the same time, it can also provide complete information on the hair follicle tissue structure and hair follicle blood vessels without destroying the integrity of the hair follicles and cells.

[0051] In some embodiments, the cleaning solution is PBS buffer, and the cleaning time is 0.5 to 1 min. The PBS buffer is composed of double distilled water and the following components: 8.18 g / L sodium chloride, 0.20 g / L potassium chloride, 1.42 g / L disodium hydrogen phosphate, 0.245 g / L potassium dihydrogen phosphate, and pH 7.3.

[0052] In the detection method provided by the present invention, NBT / BCIP staining solution is used to detect the blood supply around the hair follicles, and hematoxylin staining is used to detect the hair follicle structure, state and the number of hair follicle cells; the hair follicle structure and state include at least one of the length and thickness of the hair follicle, the size of the dermal papilla, the thickness of the bulge, the size of the hair bulb and the melanin content of the hair shaft; the hair follicle cells include one or more of the hair follicle bulge stem cells, the dermal papilla cells or the hair matrix cells.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] 1) The hair follicle detection kit of the present invention detects the morphology of hair follicles, the blood supply around the hair follicles, and the composition of hair follicle cells, and analyzes the health status of hair follicles from multiple dimensions, providing support for subsequent evaluation of the effects of hair follicle transplantation and targeted treatment.

[0055] 2) After being treated with the hair follicle detection kit of the present invention, the hair follicle structure and cell morphology can be directly observed under a microscope, which not only significantly shortens the detection time, but also does not destroy the integrity of the hair follicles and cells, and can provide complete information on the hair follicle tissue structure and hair follicle blood vessels. The hair follicle detection kit of the present invention only takes half an hour to complete the detection of hair follicles and issue a detection report, which is suitable for rapid detection before hair follicle transplantation, greatly shortens the traditional detection time, saves manpower and time costs and expensive antibody reagent costs, and provides a convenient and fast detection method for hair follicle detection.

[0056] 3) Compared with the traditional blood vessel detection method, the hair follicle blood supply detection kit of the present invention uses Triton X-100 or a combination of Triton X-100 and type I collagenase to increase the permeability and light transmittance of the hair follicles, which is more conducive to the penetration of the dye solution into the tissue. The effect of hair follicle blood vessel dyeing is better, and the distribution of blood vessels and the structure of the blood vessel lumen can be clearly seen, thereby knowing whether the hair follicle blood supply is sufficient.

[0057] 4) The hair follicle detection kit of the present invention is first treated with tissue treatment solution, and then blood vessel staining and hematoxylin staining are performed in sequence; or the combined treatment and blood vessel staining steps are combined and then hematoxylin staining is performed. The staining effect is relatively clear. The latter can shorten the staining time and achieve the purpose of faster detection. At the same time, through the acquisition of images at different levels, the size of the hair bulb, the dermal papilla and blood supply of the hair follicle bulb, and the hair follicle stem cells and melanin content of the bulge can be obtained, so as to analyze whether the entire hair follicle is in a healthy state. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The results of rapid staining of C57 mouse hair follicles using the method of Example 4 are shown. Figure 1 A is the dyeing result of the hair bulb; Figure 1 B is the staining result of blood vessels around hair follicles; Figure 1 C is the staining result of the bulge and hair shaft. Figure 1 D is the staining result of the bulge and hair follicle stem cells;

[0059] Figure 2 Shows the dyeing photos of hair follicle stem cells. Figure 2 A is the immunohistochemical staining photo of hair follicle stem cells; Figure 2 B is a quick staining photo of hair follicle stem cells;

[0060] Figure 3 The results of rapid staining of C57 mouse hair follicles using the method of Example 5 are shown. Figure 3 A is the dyeing result of the hair bulb; Figure 3 B is the staining result of blood vessels around hair follicles; Figure 3 C is the staining result of the bulge and hair shaft; Figure 3 D is the staining result of the bulge and hair follicle stem cells;

[0061] Figure 4 1 shows a front view of the hair follicle detection device, wherein 1-marking area, 2-first bracket, 3-second bracket, 4-sample detection area, 5-clamping area;

[0062] Figure 5 A stereoscopic diagram of a device for hair follicle detection is shown; wherein, 1-marking area, 2-first bracket, 3-second bracket, 5-clamping area, 6-cavity, 7-slot;

[0063] Figure 6 1 shows a side view of the hair follicle detection device, 1-marking area, 5-clamping area, 6-cavity, 7-slot, 8-detection slide;

[0064] Figure 7 8- detection slide,

[0065] Figure 8 801 is an upper slide, 802 is a lower slide;

[0066] Fig. 9 The NBT / BCIP staining results of C57 mouse hair follicles are shown. Fig. 9 AD are the NBT / BCIP staining results after treatment for 5, 10, 15, and 30 min, respectively;

[0067] Fig.10 The NBT / BCIP staining results of C57 mouse hair follicles are shown. Fig.10 A is the staining result of Triton X-1000 0.05% (v / v) + type I collagenase 0.1% (w / w); Fig.10 B is the staining result of Triton X-1000 0.1% (v / v) + type I collagenase 0.1% (w / w); Fig.10 C is the staining result of Triton X-1000 0.3% (v / v) + type I collagenase 0.1% (w / w); Fig.10 D is the staining result of Triton X-1000.5% (v / v) + type I collagenase 0.1% (w / w); Fig.10 E is the staining result of Triton X-100 1% (v / v) + collagenase type I 0.1% (w / w); Fig.10F is the staining result of Triton X-1000.3% (v / v);

[0068] Fig.11 The results of NBT / BCIP staining and hair follicle cell staining of C57 mice are shown. Fig.11 A is the staining result of Triton X-1000 0.1% (v / v) + type I collagenase 0.01% (w / w); Fig.11 B is the staining result of Triton X-1000 0.1% (v / v) + type I collagenase 0.1% (w / w); Fig.11 C is the staining result of Triton X-1000 0.1% (v / v) + type I collagenase 0.5% (w / w); Fig.11 D is the staining result of Triton X-1000 0.1% (v / v) + type I collagenase 1% (w / w); Fig.11 E is the staining result of Triton X-1000 0.3% (v / v) + type I collagenase 0.01% (w / w); Fig.11 F is the staining result of Triton X-1000 0.3% (v / v) + type I collagenase 0.1% (w / w); Fig.11 G is the staining result of Triton X-1000 0.3% (v / v) + type I collagenase 0.5% (w / w); Fig.11 H is the staining result of Triton X-1000 0.3% (v / v) + type I collagenase 1% (w / w);

[0069] Fig.12 The invention shows that the hair follicle detection device of the present invention is used to detect hair follicles;

[0070] Fig.13 The results of hair follicles of C57 mice obtained by HE staining are shown;

[0071] Fig.14 Figure 4 shows the results of immunohistochemical staining of CD31 in the hair bulb of C57 mice. DETAILED DESCRIPTION

[0072] The present invention provides a hair follicle detection kit, method and application thereof. A person skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0073] The reagents, instruments, materials and equipment used in the present invention can all be purchased from the market.

[0074] The hematoxylin stock solution is a commercially available hematoxylin staining solution. Currently, all commercially available hematoxylin staining solutions can be used for staining in the present invention, for example, Harris hematoxylin staining solution (Zhuhai Beso Company, etc.), Cole's hematoxylin staining solution (Golden Clone Biotechnology, etc.), Mayer hematoxylin staining solution (Nanjing Shenghang Biology, etc.), modified Lillie-Mayer hematoxylin staining solution (Solarbio, etc.), Ehrlich hematoxylin staining solution (Shanghai Ruji Biology, etc.), Carazzi hematoxylin staining solution (Nanjing Senbeijia Biology, etc.), Gill modified hematoxylin staining solution (Beijing Regen Biology, etc.). 100% hematoxylin is the hematoxylin stock solution.

[0075] The preparation method of the Triton X-100 solution in the hair follicle treatment solution of the present invention is as follows: 10 μL, 20 μL, 30 μL, 40 μL or 50 μL of Triton X-100 is dissolved in 10 ml of 0.01 mol / L PBS buffer to obtain a PBS solution with a volume concentration of 0.1%, 0.2%, 0.3%, 0.4% or 0.5% Triton X-100.

[0076] The preparation method of the type I collagenase solution in the hair follicle treatment solution of the present invention is as follows: 5 mg, 10 mg, 20 mg, 30 mg, 40 mg or 50 mg of type I collagenase is respectively dissolved in 10 ml of 0.01 mol / L PBS buffer to obtain PBS solutions with a mass concentration of 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% type I collagenase.

[0077] The formula of the PBS buffer solution and the PBS cleaning solution of the present invention are both 8.18 g / L sodium chloride, 0.20 g / L potassium chloride, 1.42 g / L disodium hydrogen phosphate, 0.245 g / L potassium dihydrogen phosphate, and an aqueous solution with a pH of 7.3;

[0078] The blood vessel staining solution of the present invention adopts NBT / BCIP staining solution, and NBT / BCIP is the English abbreviation of (nitro blue tetrazolium / 5-bromo-4-chloro-3-indolyl-phosphate).

[0079] The present invention will be further described below in conjunction with embodiments:

[0080] Example 1 Hair follicle treatment solution of the present invention

[0081] The hair follicle treatment solution is composed of:

[0082] 0.3% Triton X-100 (v / v), the solvent is PBS buffer.

[0083] Example 2 Hair follicle treatment solution of the present invention

[0084] The hair follicle treatment solution is composed of:

[0085] 0.3% Triton X-100 (v / v), 0.1% type I collagenase (w / w), and the solvent was PBS buffer.

[0086] Example 3 Hair follicle treatment solution of the present invention

[0087] The hair follicle treatment solution is composed of:

[0088] 0.1% Triton X-100 (v / v), 0.1% type I collagenase (w / w), and the solvent is PBS buffer.

[0089] Example 4 Hair follicle detection kit and detection method of the present invention

[0090] The hair follicle detection kit contains the following components: 0.3% (v / v) Triton X-100 + 0.1% (w / w) type I collagenase in PBS buffer (hair follicle treatment solution), NBT / BCIP in PBS solution (0.4 mg / ml NBT, 0.19 mg / ml BCIP), hematoxylin solution (hematoxylin concentration is 100%), hydrochloric acid alcohol differentiation solution (the volume percentage concentration of concentrated hydrochloric acid in hydrochloric acid alcohol is 1%), ammonia blueing solution (the mass percentage concentration of ammonia is 0.22%) and PBS cleaning solution (PBS concentration is 0.01 mol / L).

[0091] 1. Rapid detection method of in vitro hair follicles

[0092] 1) Take 6 centrifuge tubes and add one of the above-mentioned hair follicle treatment solution, NBT / BCIP staining solution, hematoxylin solution, hydrochloric acid alcohol differentiation solution, blueing solution or PBS cleaning solution to each centrifuge tube, and the added volume of each solution is 1 ml.

[0093] 2) Take out the hair follicles to be tested from the saline solution and wash them with cleaning solution for 1 minute;

[0094] 3) Treat the hair follicles with a hair follicle treatment solution for 10 minutes and clean them with a cleaning solution for 1 minute;

[0095] 4) Stain the treated hair follicles with NBT / BCIP for 10 minutes;

[0096] 5) Use hematoxylin solution to stain the hair follicles after NBT / BCIP staining for 3 minutes and wash with cleaning solution for 0.5 minutes;

[0097] 6) Place the hair follicles in the differentiation solution for 3 minutes; wash with the cleaning solution for 0.5 minutes;

[0098] 7) Place the hair follicles in the blueing solution for 2 minutes and then wash with the cleaning solution for 1 minute;

[0099] 8) Add 2 drops of glycerol to the test slide, place the hair follicles in the glycerol and seal the slide with the test slide.

[0100] 9) The sealed hair follicles were placed on a hair follicle detection device for observation and detection, and photographed using an upright optical microscope (80i, Nikon) and an image acquisition system (SPOT FLEX).

[0101] First, the hair follicles were treated with Triton X-100 + collagenase type I for 10 minutes, and then NBT / BCIP staining and hematoxylin staining were performed in sequence. After staining, not only the hair bulb, dermal papilla, bulge and hair shaft structure of the hair follicles were obvious, the nucleus of the hair follicle stem cells was clearly stained, and the lumen and three-dimensional structure of the blood vessels of the hair follicles were also clear ( Figure 1 A-1D).

[0102] 2. Detection indicators and measurement methods of hair follicles

[0103] 2.1 Detect the length and thickness of hair follicles

[0104] Hair follicle length and thickness: Place the cleaned hair follicles parallel to the ruler and measure them using the analysis software Image J1.52k (NIH);

[0105] 2.2 Hair follicle structure and status detection

[0106] Thickness of the bulge: The clearest layer of the hair shaft at the bulge is collected under a 200x optical microscope, and the straight-line distance from the hair shaft on one side to the edge of the hair follicle is measured to indicate the thickness of the bulge.

[0107] Hair bulb size: Collect the hair bulb under a 200x field of view under an optical microscope, and use the built-in measuring ruler to measure the longest length of the hair bulb.

[0108] Melanin content: The clearest layer of the hair shaft at the bulge was collected under an optical microscope at 200 times the field of view. The black hair shaft area and the optical density value within the area were measured using Image J software. The average optical density value = optical density value within the area / black hair shaft area. The average optical density value was used to represent the melanin content.

[0109] Dermal papilla: The clearest layer of melanin in the hair bulb is collected under a 200x optical microscope, and the longest length of the transparent part (similar to an ellipse) in the melanin depression area is measured to indicate the size of the dermal papilla.

[0110] 2.3 Blood supply around hair follicles

[0111] Under a 200x field of view under an optical microscope, blood vessels close to the hair bulb of the hair follicles to be tested that were stained by the hair follicle rapid staining method were collected, and the number of transversely distributed blood vessels around the hair follicles was counted.

[0112] 2.4 Hair follicle cell detection

[0113] The hair follicle stem cells, dermal papilla cells and hair matrix cells are marked by immunohistochemical staining. The positions of the hair follicle stem cells, dermal papilla cells and hair matrix cells in the hair follicles are obtained according to the results of immunohistochemical staining. The numbers of the hair follicle stem cells, dermal papilla cells and hair matrix cells in the hair follicles to be tested in the hair follicle hematoxylin rapid staining are counted according to the obtained cell position information.

[0114] The immunohistochemical staining method includes the following steps:

[0115] 1) Sampling: After tissue block sampling, it was directly placed in 4% paraformaldehyde for fixation for 24 hours and then rinsed with running water overnight.

[0116] 2) Sectioning: The tissue blocks were placed in a 30% sucrose solution for dehydration for 16 hours, then taken out and dried, and then embedded in OCT and sectioned.

[0117] 3) Fixation: Take out the frozen sections from the refrigerator and let them dry at room temperature for a while.

[0118] 4) Washing: Wash in PBS shaker at 50 r / min for 5 min × 3.

[0119] 5) Circle drawing: Blot the water around the tissue and let the slide dry. Carefully use the PAP pen to draw a circle around the tissue.

[0120] 6) Punching: Add 0.1% TritonX-100 to each slice and incubate at room temperature for 10 minutes.

[0121] 7) Washing: Wash in PBS shaker at 50 r / min for 5 min × 3.

[0122] 8) Blocking: The sections were blocked with a 2% fetal bovine serum solution (prepared in PBS) at 37°C for 1 hour. The amount of BSA was sufficient to completely immerse the tissue.

[0123] 9) Primary antibody: Aspirate the blocking buffer, place the sections in a humidified chamber and incubate with the primary antibody diluted with 2% fetal bovine serum solution at 37°C for 1 hour (dilute the antibody according to the recommended concentration and preliminary experimental results).

[0124] 10) Washing: shake off the primary antibody and wash 5 times with PBS buffer.

[0125] 11) Secondary antibody: add secondary antibody diluted in 2% BSA solution (1:1000) to the sections and incubate at 37°C for 1 hour.

[0126] 12) Washing: shake off the secondary antibody and wash with PBS buffer for 5 min × 3.

[0127] 13) Nuclear staining: Incubate with 1ug / ml DAPI working solution at room temperature for 5 minutes.

[0128] 14) Washing: Wash with PBS for 5 min × 3.

[0129] 15) Covering: Wipe the surface of the slice dry, add a drop of anti-fluorescence quenching buffer glycerol to each slice, and cover with a coverslip.

[0130] 16) Observe under a laser confocal microscope or store at -20℃ away from light.

[0131] The function of immunohistochemical staining is to mark the location information of hair follicle stem cells, dermal papilla cells and hair matrix cells in the hair follicles. The conventional immunohistochemical staining method of hair follicle stem cells is to make paraffin sections after sampling. The preparation of paraffin sections requires dehydration, wax immersion, embedding and other steps, and then sectioning. After rehydration, the hair follicle stem cell marker antibody SOX9 is used to mark them through immunohistochemistry. It takes at least four days to obtain such results. Figure 2 The results of A can only obtain hair follicle stem cells at a certain level; the present invention only needs to perform enzyme substrate reaction staining once, and the subsequent detection of hair follicle cells only needs rapid staining method to detect hair follicle cells through rapid staining, which only takes more than ten minutes, and the number and status of all hair follicle stem cells in the entire hair follicle can be seen ( Figure 2 B).

[0132] 2.5 Evaluation of the health status of hair follicles to be tested

[0133] Using the index range of normal hair follicles as the reference standard, the test results of the hair follicles to be tested are evaluated to determine whether the status of the hair follicles is normal.

[0134] Example 5 Hair follicle detection kit and detection method

[0135] The in vitro hair follicle detection kit includes the following reagents: 0.3% Triton X-100 (v / v) + 0.1% type I collagenase (w / w) + NBT / BCIP PBS solution (0.4 mg / ml NBT, 0.19 mg / ml BCIP), hematoxylin solution (hematoxylin concentration is 100%), hydrochloric acid alcohol differentiation solution (the volume percentage concentration of concentrated hydrochloric acid in hydrochloric acid alcohol is 1%), ammonia water blueing solution (the mass percentage concentration of ammonia is 0.22%) and PBS cleaning solution (PBS concentration is 0.01 mol / L).

[0136] 1. Rapid detection method of in vitro hair follicles

[0137] 1) Take 5 centrifuge tubes and add one of the above-mentioned 0.3% Triton X-100 (v / v) + 0.1% type I collagenase (w / w) + NBT / BCIP PBS solution, hematoxylin solution, hydrochloric acid alcohol differentiation solution, blueing solution or PBS cleaning solution to each centrifuge tube. The added volume of each solution is 1 ml.

[0138] 2) Take out the hair follicles to be tested from the saline solution and wash them with cleaning solution for 1 minute;

[0139] 3) Treat the hair follicles with 0.3% Triton X-100 (v / v) + 0.1% type I collagenase (w / w) + NBT / BCIP in PBS for 10 minutes and wash with cleaning solution for 1 minute;

[0140] 4) Use hematoxylin solution to stain the hair follicles after NBT / BCIP staining for 3 minutes and wash with cleaning solution for 0.5 minutes;

[0141] 5) Place the hair follicles in the differentiation solution for 3 minutes; wash with the cleaning solution for 0.5 minutes;

[0142] 6) Place the hair follicles in the blueing solution for 2 minutes and then wash with the cleaning solution for 1 minute;

[0143] 7) Add 2 drops of glycerol to the test slide, place the hair follicles in the glycerol and seal the slide with the test slide.

[0144] 8) The sealed hair follicles were placed on a hair follicle detection device for observation and detection, and photographed using an upright optical microscope (80i, Nikon) and an image acquisition system (SPOT FLEX).

[0145] The mixed solution of Triton X-100 + 0.1% type I collagenase (w / w) + NBT / BCIP was used for staining, and then hematoxylin was used for staining. The background after staining was lighter than that of Example 4. The hair bulb, dermal papilla, bulge and hair shaft structure of the hair follicle were more obvious after staining. The nucleus of the hair follicle stem cells was clearly stained, and the lumen and three-dimensional structure of the blood vessels of the hair follicle were clearer ( Figure 3 A-3D), and the hair follicles were treated with a mixture of Triton X-100 + type I collagenase and NBT / BCIP to further shorten the staining time.

[0146] 2. Detection indicators and measurement methods of hair follicles

[0147] The “hair follicle detection index and measurement method” of Example 5 is the same as that of Example 4.

[0148] Example 6 Hair follicle detection device

[0149] The hair follicle detection device includes a fixing device and a detection unit. The fixing device is composed of a marking area 1, a clamping area 5 and a fixing area. The fixing area is located between the marking area and the clamping area. A slot 7 is provided on the side of the marking area 1 close to the fixing area and on the side of the clamping area 5 close to the fixing area. The fixing area is composed of a first bracket 2 and a second bracket 3. The first bracket 2 and the second bracket 3 are both provided with a cavity 6 for accommodating the detection unit. The rectangle surrounded by the first bracket 2, the second bracket 3, the marking area and the clamping area is a sample detection area 4. The detection unit is a detection slide 8. See the front view, stereoscopic view and side view of the hair follicle detection device. Figures 4 to 6 .

[0150] In one or more embodiments, the detection glass slide is divided into an upper glass slide 801 or a lower glass slide 802, the hair follicles are placed between the upper glass slide 801 and the lower glass slide 802, and the detection glass slide 8 containing the hair follicles is placed in the cavity of the first bracket 2 and the second bracket 3 (see the detection glass slide Figure 7-8 ).

[0151] In one or more embodiments, the marking area 1, the clamping area 5 and the fixing area are an integrally formed structure, and the first bracket 2 or the second bracket 3 of the fixing area is composed of a left bracket and a right bracket, and the left bracket or the right bracket is detachably connected.

[0152] The device can make it easy to fix the glass slide and can detect at multiple angles under an optical microscope or a microscope. The hair follicle detection device comprises a fixing device and a detection unit.

[0153] Example 7 Vascular staining test

[0154] (I) Experiment on the effect of different staining times on the NBT / BCIP staining results of blood vessels around hair follicles

[0155] In order to investigate the effect of different staining times on the NBT / BCIP staining results, the whisker hair follicle blood vessels of C57 mice were detected at 5min, 10min, 15min and 30min, with NBT concentration of 0.4mg / ml and BCIP concentration of 0.19mg / ml.

[0156] Results Fig. 9 A-9D and Table 1, when NBT / BCIP staining was performed for 5 minutes, the staining was insufficient, the blood vessels were lightly colored, and the lumen of the hair follicle blood vessels was unclear. When NBT / BCIP staining was performed for 10 to 30 minutes, the staining effect was better, the staining was sufficient, and the lumen of the hair follicle blood vessels was clear. However, after 10 minutes of staining, the staining background became darker with time, and the contrast between the blood vessels and the background was not obvious, which affected the observation of the blood vessels. It was not only time-consuming, but also increased the time cost.

[0157] Table 1 NBT / BCIP staining results of blood vessels around hair follicles at different staining times

[0158]

[0159] (II) Experiment on the effect of different hair follicle treatment solutions on the NBT / BCIP staining results of blood vessels around hair follicles

[0160] The effects of different hair follicle treatment solutions on the staining results were investigated, and the effects of the combination of type I collagenase and different volume concentrations of Triton X-100 (0.05%, 0.1%, 0.3%, 0.5% and 1%) on the staining effect were investigated. The beard hair follicles of C57 mice were detected using the staining kit of experimental groups 5-10. The composition of the staining kit of experimental groups 5-10 is shown in Table 2. The detection method is as follows:

[0161] 1) Take out the hair follicles to be tested from the saline solution and wash them with cleaning solution for 1 minute;

[0162] 2) Treat the hair follicles with a hair follicle treatment solution for 10 minutes and then clean them with a cleaning solution for 1 minute;

[0163] 3) Use NBT / BCIP staining solution to stain the hair follicles for 10 minutes and wash with cleaning solution for 0.5 minutes;

[0164] 4) Add 2 drops of glycerol to the test slide, place the hair follicles in the glycerol and seal the slide with the test slide.

[0165] 5) The sealed hair follicles were placed on a hair follicle detection device for observation and detection, and photographed using an upright optical microscope (80i, Nikon) and an image acquisition system (SPOT FLEX).

[0166] 6) The clarity of the experimental group 5-10 after staining was evaluated using the image clarity assessment green version 0.4 software, and the vascular staining effect was scored according to the following standards: whether the color depth of the blood vessels outside the hair follicles and inside the dermal sheath was appropriate, and whether the overall vascular lumen and vascular distribution of the hair follicles were clear after staining. The evaluation was divided into four levels: A, B, C, and D. Staining result scoring standards: Grade A films: ≥90 points (excellent), Grade B films: 75-89 points (good), Grade C films: 60-74 points (basically qualified), and Grade D films: ≤59 points (unqualified).

[0167] Depend on Fig.10 As shown in A-10F and Table 2, the clarity of the stained images and the effect of blood vessel staining were better than those in Experimental Group 2, which was not treated with hair follicle treatment solution, when treated with Triton X-100 alone for 10 minutes or with Triton X-100 + type I collagenase for 10 minutes.

[0168] At the same time, the NBT / BCIP staining clarity (greater than 95%) and the blood vessel staining effect score (grade A) after treatment with 0.1% (w / w) type I collagenase + 0.1-0.3% (v / v) Triton X-100 (experimental groups 6-7) were better than the NBT / BCIP staining clarity (88.2%) and blood vessel staining effect score (grade B) after treatment with Triton X-100 (experimental group 10) alone. In addition, after treatment with type I collagenase 0.1% (w / w) + Triton X-1000 0.3% (v / v) for 10 minutes and NBT / BCIP staining for 10 minutes, the lumen of the blood vessels of the hair follicles was clearer, the blood vessel distribution could be clearly seen, the staining effect was optimal, and it was significantly better than other concentration combinations. The results showed that the combination of 0.3% Triton X-100 (v / v) + 0.1% type I collagenase (w / w) could better form appropriate porosity and openings on the hair follicle vessel wall and the hair follicle dermal sheath, which was beneficial for the uniform penetration of NBT / BCIP dye into the vessel lumen.

[0169] Table 2 Effects of different hair follicle treatment solutions on vascular staining results

[0170]

[0171] Example 8 Experiment on the influence of hair follicle perivascular staining and hair follicle cell staining experimental results

[0172] In order to simultaneously improve the staining effect of blood vessels and hair follicle cells around hair follicles, Triton X-100 solution + type I collagenase were used in combination to investigate the effects of different ratios of Triton X-100 and + type I collagenase on the staining effect of blood vessels and hair follicle cells of C57 mice. The composition of the hair follicle detection kit for experimental groups 11-18 is shown in Table 3, and the detection method for experimental groups 11-18 is as follows:

[0173] 1) Take out the hair follicles to be tested from the saline solution and wash them with cleaning solution for 1 minute;

[0174] 2) Treat the hair follicles with a hair follicle treatment solution for 10 minutes and then clean them with a cleaning solution for 1 minute;

[0175] 3) Use NBT / BCIP staining solution to stain the hair follicles for 5 or 10 minutes, and wash with cleaning solution for 0.5 minutes;

[0176] 4) Use hematoxylin solution to stain the hair follicles after NBT / BCIP staining for 3 minutes and wash with cleaning solution for 0.5 minutes;

[0177] 5) Place the hair follicles in the differentiation solution for 3 minutes; wash with the cleaning solution for 0.5 minutes;

[0178] 6) Place the hair follicles in the blueing solution for 2 minutes and then wash with the cleaning solution for 1 minute;

[0179] 7) Add 2 drops of glycerol to the test slide, place the hair follicles in the glycerol and seal the slide with the test slide.

[0180] 8) The sealed hair follicles were placed on a hair follicle detection device for observation and detection, and photographed using an upright optical microscope (80i, Nikon) and an image acquisition system (SPOT FLEX).

[0181] 9) Evaluation method: According to the HE staining standard of "Clinical Technical Operation Specifications. Pathology Volume", with other items unchanged, the evaluation is divided into four levels: A, B, C, and D according to item 7 "good tissue transparency" and item 8 "clear contrast between nucleus and cytoplasm" and whether the blood vessel staining is clear. Staining result scoring standard: Grade A film: ≥90 points (excellent), Grade B film: 75-89 points (good), Grade C film: 60-74 points (basically qualified), Grade D film: ≤59 points (unqualified).

[0182] Table 3 Staining results of blood vessels and hair follicle cells around hair follicles by different hair follicle treatment solutions

[0183]

[0184] Depend on Fig.11 As shown in A-11H and Table 3, the quality analysis of the stained images after treatment with different ratios of Triton X-100 and + type I collagenase was performed. When the volume concentration of Triton X-100 was 0.1% or 0.3%, the mass concentration of type I collagenase was 0.01% to 0.1% (experimental groups 11, 12, 15 and 16), the hair follicle cells and hair follicle blood vessels had good staining effects (grade A or grade B), which were better than the staining effects of experimental groups 13, 14, 17 and 18. When the mass percentage concentration of type I collagenase was ≥1% (v / v%), the stained tissue had poor light transmittance, the coloring was too dark, the contrast between the cell nucleus and the cytoplasm was poor, and the blood vessel staining was unclear (grade D).

[0185] Example 9 Comparative experiment between the detection method of the present invention and the traditional staining method

[0186] The detection method and detection results (including hair follicle cells and blood vessels around hair follicles) of the present invention refer to Example 4. The mouse hair follicles are washed with physiological saline; placed in the staining reagent of Example 4 for staining; the stained tissue sample is placed on a detection slide, 90% glycerol is added to cover the tissue, and the detection slide is used for cover; the detection unit containing the tissue sample is inserted into the fixing device from the cavity on the side of the fixing device and fixed on the fixing device; the result is observed under a microscope, and the front and back sides can be turned over to observe the tissue morphology (see Fig.12 ).

[0187] (1) The conventional HE staining method is used in the hair follicle cell detection method. The experimental results are as follows Fig.12 The images were taken for conventional HE-stained sections. Since the sections of the HE method are thin, they can only show cells and structures in one plane and are not three-dimensional.

[0188] Compared with the conventional HE method, the images taken by the method and the device of the present invention on the hair follicles of C57 mice do not require slicing and can clearly display the cells and structures at different levels of the hair follicles with a three-dimensional sense (see Fig.13 ).

[0189] (2) Existing hair follicle blood vessel detection

[0190] Detection tissue: mouse hair follicles, immunohistochemical CD31 hair follicle blood vessel staining, the hair follicles need to be cleaned, embedded, sliced, then punched with Triton, blocked with 2% BSA, incubated with primary antibody overnight, then incubated with secondary antibody, and finally observed under a microscope. The test results are as follows Fig.14 As shown. The immunohistochemical CD31 staining process takes two days, and it should be noted that when embedding hair follicles, it is necessary to pay attention to the fact that the hair follicles must be parallel to the bottom of the embedding box, and bubbles are easily generated around the hair follicles. When slicing, the complete longitudinal section of the hair follicles must be cut. It is necessary to cut many slices first and constantly adjust and check with a microscope in time to determine whether this surface is cut. During the immunofluorescence staining process, due to the complex structure of the hair follicles, some tissues are easy to fall off, especially the dermal papilla part of the hair bulb, which will cause some blood vessels to be missing, and the number of blood vessels detected will be affected. Moreover, it is only the number of blood vessels in a certain section, and it is impossible to judge whether the overall blood supply of the hair follicles is normal.

[0191] Compared with immunohistochemical CD31 hair follicle vascular staining, the present invention does not require steps such as slicing when performing vascular staining, and adds Triton or a combination of Triton and type I collagenase to treat the hair follicles. After the treatment, the hair follicle vascular staining effect is better, and the vascular distribution at different levels of the hair follicles, as well as the number of blood vessels and the three-dimensional structure of the vascular lumen can be clearly and completely displayed, thereby knowing whether the blood supply to the hair follicles is sufficient.

[0192] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A hair follicle detection kit, characterized in that: The invention comprises a hair follicle treatment solution and a dyeing solution, wherein the hair follicle treatment solution is composed of Triton X-100, type I collagenase and a solvent; the dyeing solution is an NBT / BCIP dyeing solution or a combination of an NBT / BCIP dyeing solution and a hematoxylin dyeing solution; wherein the volume percentage concentration of Triton X-100 is 0.1-0.3%, and the mass percentage concentration of type I collagenase is 0.1%.

2. The hair follicle detection kit according to claim 1, characterized in that: The staining solution is a combination of NBT / BCIP staining solution and hematoxylin staining solution, and the kit also includes differentiation solution and blueing solution.

3. The hair follicle detection kit according to claim 1, characterized in that: The solvent is PBS buffer or double distilled water.

4. The hair follicle detection kit according to claim 1, characterized in that: In the NBT / BCIP staining solution, the concentration of NBT is 0.1-0.6 mg / ml, the concentration of BCIP is 0.05-0.3 mg / ml, and the solvent is PBS buffer.

5. A method for detecting in vitro hair follicles, characterized in that: The method of using the hair follicle detection kit according to any one of claims 1 to 4 to detect the hair follicles to be detected comprises the following steps: a1. Wash the hair follicles to be tested, treat them with hair follicle treatment solution for 8-20 minutes, wash them, and then stain them with NBT / BCIP staining solution for 3-15 minutes; b1. Wash the stained hair follicles and place them on a test slide, add 80-95% glycerol, seal the slide, and observe under a microscope; or, a2. Wash the hair follicles to be tested and treat them with hair follicle treatment solution for 8-20 minutes, then wash them and stain them with NBT / BCIP solution for 3-15 minutes, and then stain them with hematoxylin solution for 2-10 minutes; b2. The stained hair follicles were treated with differentiation solution and blueing solution in turn, placed on a test slide, and 80-95% glycerol was added. The slide was sealed and observed under a microscope.

6. A method for detecting in vitro hair follicles, characterized in that: The method of detecting hair follicles to be detected by using the hair follicle detection kit according to any one of claims 1 to 4 comprises: a3. After washing the hair follicles to be tested, add hair follicle treatment solution and NBT / BCIP staining solution and treat for 8-20 minutes; b3. Wash the stained hair follicles and place them on a test slide, add 80-95% glycerol, seal the slide, and observe under a microscope; or, a4. After washing the hair follicles to be tested, add hair follicle treatment solution and NBT / BCIP staining solution and treat for 8-20 minutes; b4, use hematoxylin solution to stain the hair follicles after NBT / BCIP staining for 2-10 minutes; c4. Treat the stained hair follicles with differentiation solution and blueing solution in turn, place them on a test slide, add 80-95% glycerol, seal the slide, and observe under a microscope.

7. The detection method according to any one of claims 5 to 6, characterized in that: The cleaning solution used for cleaning is PBS buffer.

8. The detection method according to any one of claims 5 to 6, characterized in that: In the detection method, NBT / BCIP staining solution is used to detect the blood supply around the hair follicles, and hematoxylin staining is used to detect the hair follicle structure, state and the number of hair follicle cells; the hair follicle structure and state include at least one of the length and thickness of the hair follicle, the size of the dermal papilla, the thickness of the bulge, the size of the hair bulb and the melanin content of the hair shaft; the hair follicle cells include one or more of the hair follicle bulge stem cells, the dermal papilla cells or the hair matrix cells.

Images