A defatting fixative and a method of preparing the same

Abstract

The application discloses a defatting fixing solution and a preparation method thereof, which comprises the following components: formaldehyde 100-200 ml / L, ethyl acetate 20-50 g / L, anhydrous ethanol 100-300 ml / L, methanol 100-300 ml / L, glycerol 50-100 ml / L, glacial acetic acid 50-100 ml / L and TEMED 5-10 g / L. The defatting fixing solution can quickly dye the sample and has good dyeing effect, can quickly dissolve and remove the fat tissue in the sample, can promote the hardening of the fiber component and lymph nodes and other tissues in the fat, and has the effects of promoting the sampling speed and the lymph node detection rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a defatting fixative and a preparation method thereof. BACKGROUND

[0002] Pathological diagnosis is the gold standard for tumor diagnosis, and it provides a very critical directional treatment basis before, during and after tumor treatment. In pathological diagnosis, the processing of excised tissues is an important factor affecting the quality of pathological diagnosis. Generally, fresh ex vivo specimens are usually fixed in formalin fixative, because formaldehyde can preserve the morphological structure of the tissue for a long time and very well. The purpose of fixation is to preserve the original morphological structure of cells and tissues, and the fixative can prevent endogenous lysosomal enzymes from autolysis of their own tissues and cells, and inhibit the growth of bacteria and molds. Fat tissue fixative is generally composed of Bouin's solution and ethanol. However, the difficulty of taking materials in the pathology department, especially lymph node materials, is greatly increased. For gastric cancer, intestinal cancer, breast cancer and other tumor radical resection specimens, thorough lymph node sampling is crucial for tumor diagnosis, treatment plan development and prognosis. However, the conventional fixative and existing specimen fixation method cannot meet the requirements of rapid fixation while improving the sampling speed and detection rate of lymph nodes, so that the pathological doctor's method of long-term ex vivo fat tissue hand-to-hand cutting type searching for lymph nodes has many drawbacks such as insufficient sampling, long operation time, and ultimately affects the patient's treatment plan, prognosis and outcome.

[0003] Fat tissue contains a large amount of oil components and cannot be completely fixed and dehydrated. Fat is prone to hardening and shrinking during the fixation process, which will seriously affect the staining effect in the subsequent staining.

[0004] In summary, the present application provides a defatting fixative which can quickly dissolve and remove fat tissue in the sample, and at the same time promote the hardening of fiber components and lymph nodes in fat, thereby promoting the sampling speed and lymph node detection rate. SUMMARY

[0005] In order to overcome the defects in the prior art, the present application provides a defatting fixative and a preparation method thereof, which has the characteristics of low cost, easy configuration and convenient use, and can quickly dissolve and remove fat tissue in the sample, and at the same time promote the hardening of fiber components and lymph nodes in fat, thereby promoting the sampling speed and lymph node detection rate. The present application discloses a defatting fixative, which comprises the following components:

[0006] formaldehyde 100-200 ml / L,

[0007] ethyl acetate 20-50 g / L,

[0008] Anhydrous ethanol 100-300 ml / L

[0009] Methanol 100-300 ml / L

[0010] Glycerin 50-100 ml / L

[0011] 50-100 ml / L of glacial acetic acid

[0012] TEMED 5~10g / L.

[0013] In some embodiments, a degreasing fixative further includes: Tween-20 1-10 mg / L and DMSO 50-100 ml / L.

[0014] This invention also discloses a method for preparing a degreasing fixative, comprising the following steps:

[0015] Take 100-200ml of formaldehyde, 20-50g of ethyl acetate, 100-300ml of anhydrous ethanol, 100-300ml of methanol, 50-100ml of glycerol, 50-100ml of glacial acetic acid and 5-10g of TEMED, mix them evenly by sonication, dilute with water to 1L, and store at 4℃ for 16h.

[0016] Optionally, 1-10 mg of Tween-20 and 50-100 ml of DMSO may also be added during the above preparation process.

[0017] Optionally, in the above preparation method, the ultrasonic power of ultrasonic mixing is 200W, the ultrasonic time is 20-25min, and the mixing temperature is room temperature. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows the effect of staining and fixing using Example 1. The image shows uniform staining, minimal residual dye, and good results.

[0020] Figure 2 The image shows the effect of staining and fixing using Example 2. The image shows good staining effect, clean degreasing, less residual dye, and uniform staining overall.

[0021] Figure 3 The image shows the effect of staining and fixing using Example 3. The image shows uniform staining, minimal residual dye, and good results.

[0022] Figure 4 The fixed effect picture dyed with Example 4 shows that the dyeing is uniform, residual dye is less, and the effect is good.

[0023] Figure 5 The fixed effect picture dyed with Example 5 shows that the dyeing is uniform, residual dye is less, and the effect is very good.

[0024] Figure 6 The fixed effect picture dyed with Comparative Example 1 shows that the dyeing is not uniform, residual dye is less, and the effect is poor.

[0025] Figure 7 The fixed effect picture dyed with Comparative Example 2 shows that the dyeing is not uniform, and the degreasing is not clean. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below. The specific conditions not noted in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted by the manufacturers are all conventional products which can be obtained by market purchase.

[0027] The features and performances of the present application will be further described in detail below in combination with the embodiments.

[0028] Example 1 Preparation of degreasing fixing solution:

[0029] Take 100ml of formaldehyde, 20g of ethyl acetate, 100ml of anhydrous ethanol, 100ml of methanol, 50ml of glycerol, 50ml of glacial acetic acid and 5g of TEMED in a 2L beaker, mix uniformly under ultrasonic, the ultrasonic power is 200W, and the ultrasonic time is 20min. After ultrasonic, stand and dilute to 1L with a volumetric flask. The prepared degreasing fixing solution is stored at 4℃ for 16h.

[0030] Lymphoid tissue fixation:

[0031] After the colon tissue is taken, the degreasing fixing solution is added, the volume of the degreasing fixing solution added is 10 times of the colon tissue, and then the fixing is carried out at room temperature for 3h;

[0032] The fixed colon tissue is placed in a full-automatic dehydration machine for treatment, treated with 75wt% ethanol for 1.5h, then treated with xylene for transparency for 30min, and then treated with melted paraffin for wax immersion. Then, slicing is carried out.

[0033] HE staining, after the paraffin section is dewaxed, it is dyed with hematoxylin for 5 minutes, and the free hematoxylin is washed with running water. 5% acetic acid is differentiated for 1 minute, the acetic acid is washed away with running water, eosin is dyed for 1 minute, and the free eosin is washed clean with running water, and 70%, 80%, 90% and 100% ethanol is dehydrated for 10 seconds in turn, and finally, xylene is treated for 1 minute.

[0034] Sectioning, the dyed section is dripped with neutral gum, and the section is sealed. The staining effect is observed under a microscope. From the above Figure 1 , it can be seen that the lymphatic tissue has good staining effect, less residual dye, and overall uniform staining.

[0035] Example 2

[0036] Formaldehyde 150ml, ethyl acetate 30g, anhydrous ethanol 200ml, methanol 300ml, glycerol 70ml, glacial acetic acid 60ml, TEMED 7g are taken in a 2L beaker, ultrasonic mixing is uniform, the ultrasonic power is 200W, and the ultrasonic time is 20 minutes. After ultrasonic, it is placed and diluted to 1L with a volumetric flask. The prepared defatting fixative is stored at 4℃ for 16 hours.

[0037] Lymphatic tissue fixation:

[0038] After the omental tissue is taken, the defatting fixative is added, the volume of the added defatting fixative is 10 times that of the colon tissue, and then the omental tissue is fixed at room temperature for 3 hours;

[0039] The fixed omental tissue is placed in a full-automatic dehydration machine, treated with 75wt% ethanol for 1.5 hours, then treated with xylene for transparency for 30 minutes, and then treated with melted paraffin for wax immersion. Then sectioning is performed.

[0040] HE staining, after the paraffin section is dewaxed, it is dyed with hematoxylin for 5 minutes, and the free hematoxylin is washed with running water. 5% acetic acid is differentiated for 1 minute, the acetic acid is washed away with running water, eosin is dyed for 1 minute, and the free eosin is washed clean with running water, and 70%, 80%, 90% and 100% ethanol is dehydrated for 10 seconds in turn, and finally, xylene is treated for 1 minute.

[0041] Sectioning, the dyed section is dripped with neutral gum, and the section is sealed. The staining effect is observed under a microscope. From the above Figure 2 , it can be seen that the tissue has good staining effect, clean defatting, less residual dye, and overall uniform staining.

[0042] Example 3

[0043] Take formaldehyde 100ml, ethyl acetate 50g, anhydrous ethanol 300ml, methanol 150ml, glycerol 100ml, glacial acetic acid 100ml, TEMED 10g in a 2L beaker, ultrasonic mixing uniform, ultrasonic power 200W, ultrasonic time 20min. After ultrasonic, stand with volumetric flask to constant volume to 1L. The prepared defatting fixative is stored at 4℃ environment for 16h.

[0044] Lymphoid tissue fixation:

[0045] After the breast axillary tissue is taken, the defatting fixative is added, and the volume of the added defatting fixative is 10 times that of the colon tissue, and then it is fixed at room temperature for 3h;

[0046] The fixed breast axillary tissue is placed in a full-automatic dehydration machine for processing, treated with 75wt% ethanol for 1.5h, then treated with xylene for transparency for 30min, and then treated with melted paraffin for wax immersion. Then slice.

[0047] HE staining, after the above paraffin section is dewaxed, stained with hematoxylin for 5min, and the free hematoxylin is washed with running water. 5% acetic acid differentiation for 1min, wash away the acetic acid with running water, eosin staining for 1min, and wash away the free eosin with running water, dehydrate with 70%, 80%, 90% and 100% ethanol for 10s, and finally treat with xylene for 1min.

[0048] Sectioning, the stained section is dropped with neutral gum, and the section is sealed. It is observed under a microscope to observe the staining effect. From Figure 3 the lymphoid tissue staining effect is good, and the residual dye is less, and the overall staining is uniform.

[0049] Example 4

[0050] Take formaldehyde 100ml, ethyl acetate 20g, anhydrous ethanol 100ml, methanol 100ml, glycerol 50ml, glacial acetic acid 50ml, TEMED 5g, Tween-20 1 and DMSO 100ml in a 2L beaker, ultrasonic mixing uniform, ultrasonic power 200W, ultrasonic time 20min. After ultrasonic, stand with volumetric flask to constant volume to 1L. The prepared defatting fixative is stored at 4℃ environment for 16h.

[0051] Lymphoid tissue fixation:

[0052] After the colon tissue is taken, the defatting fixative is added, and the volume of the added defatting fixative is 10 times that of the colon tissue, and then it is fixed at room temperature for 3h;

[0053] The fixed colon tissue is placed in a full-automatic dehydration machine for treatment, 75wt% ethanol is used for treatment, dehydration time is 1.5h, then xylene is used for transparent treatment, treatment time is 30min, then melted paraffin is used for wax immersion treatment. Then slicing is performed.

[0054] HE staining, after dewaxing treatment of the above paraffin section, hematoxylin staining is performed for 5min, free hematoxylin is washed with running water. 5% acetic acid differentiation is performed for 1min, acetic acid is washed away with running water, eosin staining is performed for 1min, free eosin is washed away with running water, 70%, 80%, 90% and 100% ethanol is used for dehydration for 10s, finally xylene treatment is performed for 1min.

[0055] Section preparation, the stained section is dripped with neutral balsam, and then is sealed. The staining effect is observed under a microscope. From Figure 4 it can be seen that the tissue staining effect is good, residual dye is less, and the overall staining is uniform.

[0056] Example 5

[0057] Formaldehyde 100ml, ethyl acetate 20g, anhydrous ethanol 100ml, methanol 100ml, glycerol 50ml, glacial acetic acid 50ml, TEMED 5g, Tween-20 10mg and DMSO 50ml are taken in a 2L beaker, ultrasonic mixing is uniformly performed, ultrasonic power is 200W, ultrasonic time is 20min. After ultrasonic treatment, the solution is left to stand and is diluted to 1L with a volumetric flask. The prepared defatted fixative is stored at 4°C for 16h.

[0058] Lymphoid tissue fixation:

[0059] After the colon tissue is taken, the defatted fixative is added, the volume of the added defatted fixative is 10 times that of the colon tissue, then the colon tissue is fixed at room temperature for 3h;

[0060] The fixed colon tissue is placed in a full-automatic dehydration machine for treatment, 75wt% ethanol is used for treatment, dehydration time is 1.5h, then xylene is used for transparent treatment, treatment time is 30min, then melted paraffin is used for wax immersion treatment. Then slicing is performed.

[0061] HE staining, after dewaxing treatment of the above paraffin section, hematoxylin staining is performed for 5min, free hematoxylin is washed with running water. 5% acetic acid differentiation is performed for 1min, acetic acid is washed away with running water, eosin staining is performed for 1min, free eosin is washed away with running water, 70%, 80%, 90% and 100% ethanol is used for dehydration for 10s, finally xylene treatment is performed for 1min.

[0062] Section preparation, the stained section is dripped with neutral balsam, and then is sealed. The staining effect is observed under a microscope. From Figure 5As can be seen, the staining effect of lymphoid tissue is very good, the staining is uniform, and the results are accurate and clear.

[0063] Comparative Example 1

[0064] Prepare a 37% formaldehyde formalin solution.

[0065] Lymphatic tissue fixation:

[0066] After obtaining colon tissue, a defatting fixative was added, with the volume of the defatting fixative being 10 times that of the colon tissue, and then fixed at room temperature for 6 hours.

[0067] The fixed colon tissue was processed in an automated dehydrator using 75 wt% ethanol for 1.5 hours, followed by clearing with xylene for 30 minutes, and then impregnation with molten paraffin. The tissue was then sectioned.

[0068] HE staining: After dewaxing the paraffin sections, stain with hematoxylin for 5 min, then rinse with running water to remove free hematoxylin. Differentiate with 5% acetic acid for 1 min, then rinse with running water to remove the acetic acid. Stain with eosin for 1 min, then rinse with running water to remove free eosin. Dehydrate with 70%, 80%, 90%, and 100% ethanol for 10 s in sequence, and finally treat with xylene for 1 min.

[0069] Prepare slides: Apply neutral resin to the stained sections and mount. Observe the staining effect under a microscope. Figure 6 As can be seen, the staining effect of lymphoid tissue is very poor, the staining is uneven, and the lymphoid tissue is unclear and difficult to identify.

[0070] Comparative Example 2

[0071] Take 100ml formaldehyde, 20g ethyl acetate, 100ml anhydrous ethanol, 100ml methanol, 50ml glycerol, and 50ml glacial acetic acid and place them in a 2L beaker. Sonicate the mixture thoroughly at 200W for 20 minutes. After sonication, allow it to stand and then dilute to 1L using a volumetric flask. Store the prepared defatting and fixing solution at 4℃ for 16 hours.

[0072] Lymphatic tissue fixation:

[0073] After the greater omentum tissue was harvested, a defatting fixative was added. The volume of the defatting fixative added was 10 times that of the colon tissue, and then it was fixed at room temperature for 3 hours.

[0074] The fixed greater omentum tissue was placed in an automated dehydrator and treated with 75 wt% ethanol for 1.5 hours. It was then cleared with xylene for 30 minutes, followed by paraffin infiltration with molten paraffin. Finally, it was sectioned.

[0075] HE staining, after the paraffin section is dewaxed, it is dyed with hematoxylin for 5 min, and the free hematoxylin is washed with running water. 5% acetic acid is differentiated for 1 min, the acetic acid is washed away with running water, eosin is dyed for 1 min, and the free eosin is washed clean with running water, and then 70%, 80%, 90% and 100% ethanol is used for dehydration for 10 s, and finally, xylene is used for treatment for 1 min.

[0076] Preparation of tablets, the dyed section is dripped with neutral gum, and the section is sealed. The staining effect is observed under a microscope. From the above, it can be seen that more fat tissue is left, resulting in uneven staining, inaccurate results and poor staining effect. Figure 6

[0077] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A defatting fixative solution, characterized in that, It comprises the following ingredients: Formaldehyde 100-200ml / L, Ethyl acetate 20-50g / L, Anhydrous ethanol 100-300ml / L, Methanol 100-300ml / L, Glycerol 50-100ml / L, Glacial acetic acid 50-100ml / L, TEMED 5-10g / L.

2. The deparaffinizing fixative of claim 1, wherein, It also comprises the following ingredients: Tween-20 1-10mg / L and DMSO 50-100ml / L.

3. A method for preparing a defatting fixative solution, characterized by, The preparation method comprises the following steps: Take formaldehyde 100-200ml, ethyl acetate 20-50g, anhydrous ethanol 100-300ml, methanol 100-300ml, glycerol 50-100ml, glacial acetic acid 50-100ml and TEMED 5-10g, ultrasonic mix uniformly, use water to constant volume to 1L, and store at 4℃ environment for 16h.

4. The method for preparing a deparaffinized fixative solution according to claim 3, characterized in that, Further add Tween-20 1-10mg and DMSO 50-100ml.

5. The method for preparing a deparaffinized fixative solution according to claim 3 or 4, characterized in that, The ultrasonic mixing ultrasonic power is 200W, the ultrasonic time is 20-25min, and the mixing temperature is room temperature.

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