Improved renal biopsy paraffin section antigen repair method and antigen repair liquid

Abstract

The invention provides an antigen repair liquid. The antigen repair liquid is composed of 50-200 ml of a TBS buffer solution, 0.02-0.3 g of trypsin and 0.01-0.5 g of anhydrous calcium chloride. The invention also provides a renal biopsy paraffin section antigen repair method, which comprises the following steps: dewaxing and washing a paraffin section by using a volatile organic solvent to obtain a dewaxed section, putting the dewaxed section into the antigen repair liquid, treating at 35-40 DEG C for 0.5-2 hours, dropwise adding the confining liquid after washing, and keeping the temperature at 35-40 DEG C for 5-20 minutes. The device is good in safety, and experimental accident risks such as container bursting and scalding are reduced; the dissection rate is low, and repeated experiments and specimen loss caused by tissue dissection are reduced; the dyeing effect is stable and remarkable, the reproducibility is good, and the dyeing quality is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antigen retrieval of kidney biopsy tissue sections, and particularly relates to an improved antigen retrieval method for kidney biopsy paraffin sections based on enzyme retrieval and an antigen retrieval solution. BACKGROUND

[0002] Kidney tissue immunofluorescence can use frozen sections or paraffin sections, and kidney tissue immunohistochemistry often uses paraffin sections. The advantages of paraffin sections are that they can be stored for a long time, and kidney biopsy specimens from many years ago can be used for relevant immunofluorescence or immunohistochemical staining for the purpose of revising diagnosis and retrospective research; they are convenient for transportation, and primary units can transport the sections to superior hospitals for diagnosis and consultation; they can be fixed by formaldehyde to show clearer tissue structure and cell morphology; and they have longer immunofluorescence quenching time, which is conducive to result interpretation. Kidney tissue immunofluorescence often uses frozen tissue sections, and the limitations of frozen tissue are that sometimes there are no glomeruli or only sclerotic glomeruli, which cannot be diagnosed; frozen section machines are expensive, and it is difficult for primary units to develop them, and frozen sections are not easy to store and transport; sometimes the antigens of frozen sections are covered, and antigenic determinants need to be exposed after further paraffin section antigen retrieval (AR) to improve the positive rate of staining. Therefore, preparing paraffin sections for kidney tissue immunofluorescence and immunohistochemical staining has great value for kidney biopsy histopathological diagnosis.

[0003] In the fixation step of kidney tissue paraffin section preparation, formaldehyde or paraformaldehyde can cause protein cross-linking or blocking of part of the antigens in the tissue, change the spatial structure of the antigenic determinant epitope, i.e., the antigenic determinant is covered or forms a hidden antigenic determinant, loses the antigenic immunological activity, causes the antibody to be unable to fully combine with the antigen, and causes false negatives of immunofluorescence or immunohistochemistry. Antigen retrieval can reverse the cross-linking effect of formalin, expose the antigenic determinants of the tissue, and improve the positive rate of paraffin section immunofluorescence and immunohistochemistry, and is therefore an important step in kidney pathological diagnosis.

[0004] Traditional antigen retrieval uses heat retrieval or high-pressure retrieval methods, including microwave heating antigen retrieval, high-pressure water bath antigen retrieval, and special steamer antigen retrieval. However, heat retrieval or high-pressure retrieval needs to use high temperature or high pressure conditions, and dangerous liquids such as HCl, and in addition to the high risk coefficient of the experiment, there are the following limitations: ① The tissue sections are damaged more seriously, and the tissue may be detached in the intense reaction, which needs to be repeated, and the valuable tissue specimens are wasted; ② The glass container is easy to burst, which not only affects the progress of the experiment, but also may cause personal injury to the experimental operator; ③ The two antigen retrieval methods are relatively strong, and may cause false positives in subsequent staining. The traditional enzyme retrieval method has the following limitations: ① The staining quality is low, and the false negative rate is high; ② The staining effect is unstable. SUMMARY

[0005] The present application aims to provide an improved antigen retrieval method for kidney biopsy paraffin sections based on enzyme retrieval method and an antigen retrieval solution to overcome the above-mentioned defects in the prior art.

[0006] The present application is implemented by using the following technical solutions:

[0007] An antigen retrieval solution is composed of TBS buffer 50-200 ml, trypsin 0.02-0.3 g and anhydrous calcium chloride 0.01-0.5 g.

[0008] The TBS buffer is composed of sodium chloride 137 mM / L, potassium chloride 2.7 mM / L and tris(hydroxymethyl)aminomethane (Tris, CAS: 77-86-1) 25 mM / L. The TBS buffer can be prepared by using conventional methods in the art, for example: weigh sodium chloride 137 mM, potassium chloride 2.7 mM and Tris 25 mM, dissolve in 800 mL distilled water, adjust the pH to 7.2-7.6 with HCl, and then add distilled water to 1 L. Of course, commercially available products can also be used directly.

[0009] Both trypsin (CAS: 9002-07-7, EC: 3.4.21.4) and anhydrous calcium chloride (CAS: 10043-52-4) are conventional commercially available products, and there is no specific specification limit.

[0010] Preferably, the antigen retrieval solution is composed of TBS buffer 75-150 ml, trypsin 0.05-0.2 g and anhydrous calcium chloride 0.05-0.3 g.

[0011] More preferably, the antigen retrieval solution is composed of TBS buffer 90-120 ml, trypsin 0.08-0.15 g and anhydrous calcium chloride 0.08-0.15 g.

[0012] An antigen retrieval method for kidney biopsy paraffin sections includes the following steps:

[0013] S1, using a volatile organic solvent to dewax the paraffin section;

[0014] S2, water washing to remove residual paraffin and organic solvent to obtain a dewaxed section;

[0015] S3, taking 50-200 ml of TBS buffer, adding 0.02-0.3 g of trypsin and 0.01-0.5 g of anhydrous calcium chloride, and preheating at 35-40℃ for 5-20 minutes to obtain the antigen retrieval solution;

[0016] S4, placing the dewaxed section in the antigen retrieval solution and treating at 35-40℃ for 0.5-2 hours;

[0017] S5, taking out the treated dewaxed section, and washing the residual repair solution with a PBS solution;

[0018] S6, adding a blocking solution dropwise, and incubating at 35-40℃ for 5-20 minutes.

[0019] As a preferred technical solution, the treated dewaxed section is first placed in a hydrogen peroxide-methanol solution before water washing in step S2, and incubated at 35-40℃ for 5-20 minutes. The main function of this step is to remove endogenous peroxidase in the tissue, which can reduce various staining background colors and reduce the probability of false positives.

[0020] The volatile organic solvent can be selected from common solvents in the art, for example but not limited to xylene reagent.

[0021] In step S3, the addition amount of TBS buffer is preferably 75-150 ml, and more preferably 90-120 ml; the addition amount of trypsin is preferably 0.05-0.2 g, and more preferably 0.08-0.15 g; and the addition amount of anhydrous calcium chloride is preferably 0.05-0.3 g, and more preferably 0.08-0.15 g.

[0022] In step S6, the blocking solution is an immunofluorescence blocking solution commonly used in the art.

[0023] Compared with the prior art, the present application has the following advantages:

[0024] 1. Compared with the hot repair or high-pressure repair method, the safety is good, and the risk of experimental accidents such as container explosion and scalding is reduced; the section loss is low, and the repeated experiments and specimen loss caused by tissue section loss are reduced.

[0025] 2. Compared with the conventional enzyme repair method, the staining effect of the present application is more stable and more significant, the reproducibility is good, and the staining quality is high.

[0026] The present application is further described below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Immunofluorescence images of different indexes of kidney biopsy paraffin sections repaired by the antigen repair method of the present application.

[0028] Figure 2 IgA immunofluorescence images of kidney biopsy paraffin sections repaired by the conventional antigen repair method. DETAILED DESCRIPTION

[0029] The following description is provided to enable any person skilled in the art to practice the present application. The preferred embodiments described herein are only examples of the present application and various modifications can be made by those skilled in the art without departing from the spirit and scope of the present application. The present application defined in the following description is not limited to the preferred embodiments described herein and can be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the present application.

[0030] The instruments or raw materials in the present application are not specified by the manufacturer, and are all conventional commercial instruments or raw materials. Among them, the TBS buffer adopts 20X TBS Buffer of Shengong Biotech, product number B548105-0500; trypsin adopts the product of National Pharmaceutical Group Chemical Reagent Co., Ltd.; anhydrous calcium chloride powder adopts the product of Shanghai Lingfeng Chemical Reagent Co., Ltd.; blocking solution adopts QuickBlock immunofluorescence blocking solution (P0260) of Biyun Tian Company.

[0031] The detection indicators involved in the embodiments of the present application are not mentioned, and are detected by conventional detection methods in the art.

[0032] Embodiment 1

[0033] A kind of kidney biopsy paraffin section antigen repair method, comprising the following steps:

[0034] 1. Conventional kidney biopsy paraffin section is dewaxed to water.

[0035] 2. 0.3% H202 methanol 37 DEG C incubator treatment 15 minutes (immunofluorescence staining does not need this step).

[0036] 3. Tap water is washed for 2 minutes each time, a total of 3 times.

[0037] 4. 1x100ml TBS solution is prepared, 0.1g trypsin, 0.1g anhydrous calcium chloride powder are added, and it is placed in 37 DEG C water bath, preheated for 10 minutes.

[0038] 5. The section is placed in the above-mentioned solution, and 37 DEG C water bath treatment is carried out for 1 hour.

[0039] 6. PBS solution is washed for 2 minutes each time, a total of 3 times.

[0040] 7. Drop the blocking solution and incubate in a 37 DEG C incubator for 15 minutes.

[0041] 8. PBS solution is washed for 2 minutes each time, a total of 3 times.

[0042] 9. Drop the required dyeing antibody, and incubate in a 37 DEG C incubator for 1 hour. (If secondary antibody needs to be added, first wash the primary antibody with PBS solution, then add the secondary antibody and incubate in a 37 DEG C incubator for 1 hour)

[0043] 10. Rinse with PBS solution for 2 minutes each time, three times in total.

[0044] 11. Seal the slides with glycerol and examine the slides under a microscope.

[0045] Comparative Example

[0046] Renal biopsy paraffin sections were repaired using a conventional trypsin repair method, and the pathological sections were from the same source as in Example 1. Compared to Example 1, step 4 used only TBS solution and trypsin, without the addition of anhydrous calcium chloride powder. All other procedures were the same as in Example 1. The immunofluorescence assay was the same as in Example 2, except that anhydrous calcium chloride powder was not added.

[0047] Example 2

[0048] Immunofluorescence experiments.

[0049] 1. Adjust the water bath to 37°C and wait for the water temperature to gradually rise.

[0050] 2. Stir TBS, trypsin, and anhydrous calcium chloride solution (for the comparative example, no anhydrous calcium chloride is added) in a certain ratio, place them in a 37°C water bath, and preheat for about 10 minutes.

[0051] 3. Place the dewaxed sections that require antigen retrieval in preheated antigen retrieval solution and incubate in a 37°C water bath for one hour. Note that the sections should be spaced apart and not placed too close together.

[0052] 4. Adjust the incubator to 37°C for subsequent antibody incubation.

[0053] 5. Place the sections after antigen repair and PBS solution washing on a wet box and add the antibody required for staining. Cover the wet box lid and place it in a 37℃ constant temperature box for incubation for one hour.

[0054] 6. Turn on the fluorescence microscope and fluorescence stimulator, wait for all the stimulator lights to light up, wash the incubated slices with PBS solution and seal them with glycerol, then place them on the microscope stage for viewing and filming.

[0055] The equipment used was a Leica fluorescence microscope, model: Leica DM60000B, and the software version for collecting images was LAS V4.5.

[0056] like Figure 1 As shown, the immunofluorescence experiments under different indicators of the present invention all showed very high fluorescence intensity.

[0057] Compared with the conventional trypsin repair method of the comparative example (such as Figure 2), under the same exposure intensity and gain from the pathological section of the same patient, collected by microscope camera software, although it can also be described as IgA diffusely distributed in the mesangial area of the glomerulus, only weak fluorescence can be observed, which is significantly weaker than the present application.

[0058] From the above experimental results, it can be seen that the present application is safer than the conventional thermal repair method and more effective than the conventional enzyme repair method.

[0059] The above-described embodiments are only used to illustrate the technical ideas and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot be limited to the patent application range of the present application only by the above embodiments, that is, any equivalent changes or modifications made in the spirit disclosed by the present application still fall within the patent scope of the present application.

Claims

1. An antigen repair solution, characterized in that It is composed of 50-200 ml of TBS buffer, 0.02-0.3 g of trypsin and 0.01-0.5 g of anhydrous calcium chloride.

2. The antigen retrieval solution according to claim 1, wherein The antigen retrieval solution consists of 75-150 ml of TBS buffer, 0.05-0.2 g of trypsin, and 0.05-0.3 g of anhydrous calcium chloride.

3. The antigen retrieval solution according to claim 1, wherein The antigen repair solution consists of 90-120 ml of TBS buffer, 0.08-0.15 g of trypsin and 0.08-0.15 g of anhydrous calcium chloride.

4. A method for antigen retrieval of renal biopsy paraffin sections, comprising the following steps: S1. Dewaxing the paraffin sections using volatile organic solvents; S2, washing with water to remove residual paraffin and organic solvents to obtain dewaxed sections; S3. Take 50-200 ml of TBS buffer, add 0.02-0.3 g of trypsin and 0.01-0.5 g of anhydrous calcium chloride, and preheat at 35-40°C for 5-20 minutes to obtain antigen retrieval solution; S4, placing the dewaxed sections in the antigen retrieval solution and treating at 35-40° C. for 0.5-2 hours; S5. Take out the dewaxed sections after treatment and wash them with PBS solution to remove the residual repair solution; S6. Add blocking solution dropwise and keep warm at 35-40℃ for 5-20 minutes.

5. The method according to claim 4, wherein Before washing in step S2, the treated dewaxed sections are placed in a hydrogen peroxide-methanol solution and kept warm at 35-40°C for 5-20 minutes.

6. The method according to claim 4, wherein In step S3, the amount of TBS buffer added is 75-150 ml; the amount of trypsin added is 0.05-0.2 g; and the amount of anhydrous calcium chloride added is 0.05-0.3 g.

7. The method according to claim 4, wherein In step S3, the amount of TBS buffer added is 90-120 ml; the amount of trypsin added is 0.08-0.15 g; and the amount of anhydrous calcium chloride added is 0.08-0.15 g.

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