Goose skin hair follicle microsection manufacturing and evaluating method
Through gradient ethanol dehydration and paraffin embedding technology, combined with paraformaldehyde fixation and xylene-ethanol clearing treatment, the problems of uniformity and accuracy of goose skin secondary hair follicle microscopy were solved, and a clear record of the number of hair follicles was achieved, supporting goose breeding and down quality evaluation.
Patent Information
- Application Number
- CN202510900185.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-01
- Publication Date
- 2025-09-30
AI Technical Summary
Existing technologies make it difficult to accurately assess the density and distribution of secondary hair follicles in goose skin, and the microscopic sectioning process is complex, making it difficult to ensure the uniformity of section thickness and accurate recording of the number of hair follicles.
Gradient ethanol dehydration and paraffin embedding techniques were used, combined with paraformaldehyde fixation and xylene-ethanol mixture transparency treatment to ensure the clarity of the secondary hair follicle structure and uniform slice thickness. The slice thickness and number were precisely controlled by a microtome, and HE staining was used to observe the distribution of hair follicles.
The secondary follicle structure slices are clearly sliced, easy to operate, and the number of follicles is accurately recorded, which is convenient for follicle observation and quantity analysis, and supports goose breeding and down quality evaluation.
Smart Images
Figure CN120721460A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of animal histological analysis technology and skin appendage application technology, and in particular to a method for preparing and evaluating goose skin hair follicle microsections. Background Art
[0002] The Sanhua goose is a hybrid goose breed derived from the Yangzhou white goose and one of the most popular meat goose breeds on the market. Goose down (primarily produced from secondary follicles) is the core ingredient for high-quality down products, far more valuable than the goose's vanes. Geese rely on the rich down feathers produced by secondary follicles to protect them from cold aquatic environments. The feather region of geese, particularly on the chest and abdomen, is well-developed, with numerous secondary follicles in each feather tuft, producing a high volume of high-quality down (large clusters and high loft). This down has a fluffy structure, with short, soft rachises and slender barbs without barbules. This creates a still air layer that provides insulation and warmth, making it one of the world's finest natural thermal insulation materials. The feather follicles are a unique epidermal structure, divided into primary and secondary follicles. The growth and development of these follicles significantly influences down production.
[0003] Primary and secondary follicles: At the center of each feather cluster is typically a primary follicle. This primary follicle is responsible for producing the primary, large feathers, typically the correct feathers. The correct feathers, with their distinct rachis and vanes (composed of barbs and barbules), form the primary contours of a bird's body, providing flight capabilities (flight feathers, tail feathers) or primary body coverage (coverts). Surrounding the primary follicle (usually arranged in a ring) are several smaller follicles, often called secondary follicles. They are typically responsible for producing down feathers and / or quills.
[0004] Secondary follicles typically develop after primary follicles. Down feathers produced by these secondary follicles are the most important economic feathers for geese. Their fluffy structure effectively traps air, crucial for maintaining body temperature in cold water. This is also the fundamental reason for the exceptional warmth of goose down products (down jackets and duvets). Down feathers are very fine, hair-like feathers, typically consisting of a single, slender rachis with a small number of short barbs at the tip. They may have sensory functions or help stabilize other feathers. Because goose skin is thin and has numerous hair follicles, mature techniques for preparing microscopic sections have yet to be reported. Furthermore, hair follicles grow at varying depths within the skin, making it difficult to accurately assess follicle density through microscopic sections. Summary of the Invention
[0005] The purpose of the present invention is to solve the above-mentioned problems and to propose a method for preparing and evaluating goose skin follicle microsections. The present invention provides a detailed introduction to the method for preparing and evaluating secondary hair follicle microsections of white goose skin, providing a reference for skin secondary hair follicle microsection technology. The white goose skin secondary hair follicle microsection method of the present application makes the secondary hair follicle structure slices clearly layered and easy to operate without optimizing the tissue processing process. The microsection method is used to ensure that the thickness of the secondary hair follicle slices is more uniform, and the number of primary and secondary hair follicles is uniform and accurately recorded, which facilitates the observation and quantity analysis of hair follicle slices, and plays an important role in goose breeding (especially down production) and down quality evaluation.
[0006] The purpose of the present invention can be achieved through the following technical solutions: A method for preparing and evaluating goose skin hair follicle microsections, the method specifically comprising the following steps: Step 1: Sample collection and pretreatment, this step is divided into: A1. Location and sampling: Select the skin from the back of a three-flowered goose (the area with dense secondary hair follicles), avoiding hair plucking damage, and use a scalpel to cut a 5×5mm full-thickness skin sample; A2. Rapid fixation: Immediately fix the hair follicles in 3.8%-4.2% paraformaldehyde for 24 hours to stabilize the hair follicle microstructure. Step 2: Gradient dehydration and clearing, this step is divided into: B1. Use step-by-step ethanol dehydration Perform the following operations on the slide sample with the section attached: Dehydrate the sliced samples in 75% ethanol for 8 h; Secondly, the sliced samples were dehydrated in 85% ethanol for 5 h; Secondly, the slices were dehydrated in 95% ethanol twice, each time for 2 h; Secondly, the slices were dehydrated in 100% ethanol twice, each time for 30 min; Secondly, the sliced sample was placed in a 1:1 xylene-ethanol mixture to make it transparent for 35 minutes; Secondly, the sliced samples were placed in xylene solution for over-clearing twice, with each over-clearing time of 15 minutes; Step 3: Paraffin embedding optimization, this step is divided into: C1. Place the sliced sample from step 2 into a paraffin solution with a melting point of 52-54°C for 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 56-58°C for 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 58-60°C for 2 hours; C2. Orient the hair follicle during embedding so that its longitudinal axis is perpendicular to the sectioning direction to facilitate observation of the layered structure. C3. When sampling, fix the area of the sample and cut it horizontally. When embedding, the hair shaft side is the required side. The sectioning direction is epidermis → dermis → subcutaneous tissue. C4. When trimming the slices to 5 μm thickness, press the first gear button on the microtome and cut 25-40 slices and discard them to expose the tissue surface (depending on the embedding depth, sometimes up to 40 slices may be cut and discarded); C5. Place in the refrigerator for 4-5 hours and then slice. After every 10 slices, select one to be sliced. Stop slicing when 70-90 slices have been sliced. Mark the number of slices on each slice for subsequent observation. C6. Preliminary tests were conducted with the 10mg / L group. The results showed that the primary and secondary hair follicle densities were higher between 50-60 slices. Other groups were tested with 50-60 slices, and HE staining was performed to observe and analyze the specific changes in each slice to find the slice with the largest number of hair follicles. C7. Find the optimal number of sections for staining observation and calculate the ratio of secondary hair follicles to primary hair follicles per unit area.
[0007] The further technical improvement of the present invention is: A2 in step 1, rapid fixation: immediately immersing in 4% paraformaldehyde for fixation for 24 hours to stabilize the hair follicle microstructure.
[0008] The further technical improvement of the present invention is that: in step 3, C1, the slice sample is placed in a paraffin solution with a melting point of 52-54° C. and the wax penetration time is 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 56-58°C for 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 58-60°C for infiltration, and the wax infiltration time was 2 hours.
[0009] A further technical improvement of the present invention is that: in step 3, when trimming the slices to a thickness of 5 μm, press the first gear button of the microtome, cut 30 slices and discard them, and expose the tissue exposure surface (depending on the embedding depth, sometimes up to 40 slices are cut and discarded).
[0010] A further technical improvement of the present invention is: C5 in step three, put it in the refrigerator and freeze it for 4-5 hours for slicing, select one piece for slicing after every 10 pieces are cut, and stop slicing when 80 pieces are sliced continuously, and mark the number of slices on each piece for subsequent observation.
[0011] Beneficial effects of the present invention: By selecting the back skin of the three-flowered goose to avoid damage from plucking, and using a scalpel to cut a 5×5mm full-thickness skin sample, the sample was immediately placed in 3.8%-4.2% paraformaldehyde for fixation for 24 hours, combined with step-by-step ethanol dehydration, the xylene-ethanol mixture (1:1) became transparent, which helped to reduce tissue brittleness, and the use of paraffin embedding optimization not only made the secondary follicle structure slices clearly layered and easy to operate without the need to optimize the tissue processing process, but also ensured that the thickness of the secondary follicle slices was more uniform, making the number of primary and secondary follicles uniform and accurately recorded, facilitating the observation and quantitative analysis of follicle slices, which plays an important role in goose breeding (especially down production) and down quality evaluation. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0013] Figure 1 Representative illustration of hematoxylin and eosin staining of a goose feather follicle cross section.
[0014] Figure 2 Schematic diagram of primary hair follicle density in the cross section of the abdominal hair follicles of the Sanhua goose.
[0015] Figure 3 Schematic diagram of the secondary hair follicle density in the cross section of the abdominal hair follicle of the Sanhua goose.
[0016] Figure 4 Schematic diagram of the density ratio of primary and secondary hair follicles in the cross section of the abdominal hair follicles of the Sanhua goose.
[0017] Figure 5 This is a schematic diagram of the primary hair follicle density in wax strips 52-58 of the abdominal hair follicle sections of the Sanhua goose.
[0018] Figure 6 Schematic diagram of the secondary hair follicle density in wax strips 52-58 of the abdominal hair follicle sections of the Sanhua goose. DETAILED DESCRIPTION
[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.
[0020] Experimental animals: Three-flowered goose Laboratory instruments and surgical instruments: Refrigerator, microscope, paraffin slicer, embedding box, adhesive slide, cover slip, tweezers, blade, measuring cylinder, dropper, staining jar Experimental reagents and preparation: Paraformaldehyde, ethanol solution, xylene solution, wax solution.
[0021] A method for microscopy of secondary hair follicles in white goose skin, the method specifically comprising the following steps: Step 1: Sample collection and pretreatment, this step is divided into: A1. Location and sampling: Select the skin from the back of a three-flowered goose (the area with dense secondary hair follicles), avoiding hair plucking damage, and use a scalpel to cut a 5×5mm full-thickness skin sample; A2. Rapid fixation: Immediately fix the hair follicles in 3.8%-4.2% paraformaldehyde for 24 hours to stabilize the hair follicle microstructure. Step 2: Gradient dehydration and clearing, this step is divided into: B1. Use step-by-step ethanol dehydration Perform the following operations on the slide sample with the slice attached: Dehydrate the sliced samples in 75% ethanol for 8 h; Secondly, the sliced samples were dehydrated in 85% ethanol for 5 h; Secondly, the slices were dehydrated in 95% ethanol twice, each time for 2 h; Secondly, the slices were dehydrated in 100% ethanol twice, each time for 30 min; Secondly, the sliced sample was placed in a 1:1 xylene-ethanol mixture to make it transparent for 35 minutes; Secondly, the sliced samples were placed in xylene solution for over-clearing twice, with each over-clearing time of 15 minutes; Step 3: Paraffin embedding optimization, this step is divided into: C1. Place the sliced sample from step 2 into a paraffin solution with a melting point of 52-54°C for 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 56-58°C for 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 58-60°C for 2 hours; C2. Orient the hair follicle during embedding so that its longitudinal axis is perpendicular to the sectioning direction to facilitate observation of the layered structure. C3. When sampling, fix the area of the sample and cut it horizontally. When embedding, the hair shaft side is the required side. The sectioning direction is epidermis → dermis → subcutaneous tissue. C4. When trimming the slices to 5 μm thickness, press the first gear button on the microtome and cut 30 slices and discard them to expose the tissue surface (depending on the embedding depth, sometimes up to 40 slices may be cut and discarded); C5. Place in the refrigerator for 4-5 hours and then slice. After every 10 slices, select one to be sliced. Stop slicing when 80 slices have been sliced. Mark the number of slices on each slice for subsequent observation. C6. Preliminary tests were conducted with the 10mg / L group. The results showed that the primary and secondary hair follicle densities were higher between 50-60 slices. Other groups were tested with 50-60 slices, and HE staining was performed to observe and analyze the specific changes in each slice to find the slice with the largest number of hair follicles. C7. Find the optimal number of sections for staining observation and calculate the ratio of secondary hair follicles to primary hair follicles per unit area. result:
[0022] Figure 2 The density of primary hair follicles in the cross section of the abdominal hair follicles of the Sanhua goose can be seen in the figure.
[0023] Figure 3 The density of secondary hair follicles in the cross section of the abdominal hair follicles of the Sanhua goose can be seen in the figure.
[0024] Figure 4 The density ratio of primary hair follicles to secondary hair follicles in the cross section of the abdominal hair follicles of the Sanhua goose can be seen.
[0025] Figure 5 The density of primary hair follicles can be seen in wax strips 52-58 of the abdominal hair follicle sections of the Sanhua goose.
[0026] Figure 6 The density of secondary hair follicles can be seen in the wax strips 52-58 of the abdominal hair follicle sections of the Sanhua goose. in conclusion:
[0027] Down is the goose's most important insulating layer and the core source of warmth in goose down products. Secondary follicles are the key anatomical structure that enables geese to efficiently retain heat and produce the highly economically valuable down.
[0028] The results of the biopsy showed that the number of primary and secondary follicles in the 52-58 wax strips of the abdominal hair follicles of the Sanhua goose was relatively large. This method facilitates the observation and quantitative analysis of hair follicle sections. The distribution, density and development of hair follicles are crucial for goose breeding (especially down production) and down quality assessment. The above content is merely an example and explanation of the concept of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.
Claims
1. A method for preparing and evaluating goose skin hair follicle microsections, characterized by: The method specifically comprises the following steps: Step 1: Sample collection and pretreatment, this step is divided into: A1. Location and sampling: Select the skin from the back of a three-flowered goose and cut a 5×5mm full-thickness skin sample with a scalpel; A2. Rapid fixation: Immediately fix in 3.8%-4.2% paraformaldehyde for 24 hours; Step 2: Gradient dehydration and clearing, this step is divided into: B1. Use step-by-step ethanol dehydration Perform the following operations on the slide sample with the section attached: Dehydrate the sliced samples in 75% ethanol for 8 h; Secondly, the sliced samples were dehydrated in 85% ethanol for 5 h; Secondly, the slices were dehydrated in 95% ethanol twice, each time for 2 h; Secondly, the slices were dehydrated in 100% ethanol twice, each time for 30 min; Secondly, the sliced sample was placed in a 1:1 xylene-ethanol mixture to make it transparent for 35 minutes; Secondly, the sliced samples were placed in xylene solution for over-clearing twice, with each over-clearing time of 15 minutes; Step 3: Paraffin embedding optimization, this step is divided into: C1. Perform the following operations on the sliced sample in sequence; Place the sliced sample in a paraffin solution with a melting point of 52-54°C for 1.5-2.5 hours; Secondly, the sliced sample is placed in a paraffin solution with a melting point of 56-58°C for infiltration for 1.5-2.5 hours; Secondly, the sliced sample is placed in a paraffin solution with a melting point of 58-60°C for infiltration, and the wax infiltration time is 1.5-2.5 hours; C2. Orient the embedding so that the longitudinal axis of the hair follicle is perpendicular to the sectioning direction; C3. When sampling, fix the area of the sample and cut it horizontally. When embedding, the hair shaft side is the required side. The sectioning direction is epidermis → dermis → subcutaneous tissue. C4. When trimming the slices to 5 μm thickness, press the first gear button on the microtome and cut 25-40 slices and discard them to expose the tissue surface; C5. Place in the refrigerator for 4-5 hours and slice. After every 10 slices, select one to remove. Stop slicing when 70-90 slices have been removed. Mark the number of slices removed on each slice. C6. Preliminary tests were conducted with the 10mg / L group. The results showed that the primary and secondary hair follicle densities were higher between 50-60 slices. Other groups were tested with 50-60 slices, and HE staining was performed to observe and analyze the specific changes in each slice to find the slice with the largest number of hair follicles. C7. Find the optimal number of sections for staining and observation, and calculate the ratio of secondary hair follicles to primary hair follicles per unit area.
2. The method for preparing and evaluating goose skin hair follicle microsections according to claim 1, characterized in that: A2 in step 1, quick fixation: immediately immerse in 4% paraformaldehyde for 24 hours.
3. The method for preparing and evaluating goose skin hair follicle microsections according to claim 1, characterized in that: The following operations are performed on the slice sample in C1 in step 3: The sliced samples were placed in a paraffin solution with a melting point of 52-54°C for 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 56-58°C for 2 hours; Secondly, the sliced samples were placed in a paraffin solution with a melting point of 58-60°C for infiltration, and the wax infiltration time was 2 hours.
4. The method for preparing and evaluating goose skin hair follicle microsections according to claim 1, characterized in that: In step 3, when trimming the slices to 5 μm thickness, press the first gear button on the slicer and cut 30 slices and discard them to expose the tissue surface.
5. The method for preparing and evaluating goose skin hair follicle microsections according to claim 1, characterized in that: C5 in step 3, put it in the refrigerator and freeze for 4-5 hours before slicing. After every 10 slices, select one to slice. Stop slicing when 80 slices are sliced continuously. Mark the number of slices on each slice.