A method for detecting expression of collagen type IV in renal biopsy tissue

Abstract

This invention belongs to the field of renal pathology testing technology and discloses a method for detecting type IV collagen expression in renal biopsy tissue, comprising the following steps: dewaxing and hydrating paraffin sections of renal biopsy tissue to obtain processed tissue sections; adding the processed tissue sections to a repair solution, heating to boiling, and then cooling to room temperature to obtain repaired tissue sections; adding protease to the repaired tissue sections for enzymatic digestion to obtain enzymatically digested tissue sections; adding a primary antibody to the enzymatically digested tissue sections for preliminary incubation, followed by adding a fluorescein-labeled secondary antibody against the primary antibody for further incubation. This method for detecting type IV collagen expression in renal biopsy tissue is applicable to cases where renal biopsy tissue lacks glomeruli or has too few glomeruli to detect type IV collagen expression. It has high specificity and can provide a new detection method for Alport syndrome in cases where renal biopsy tissue lacks glomeruli.

Description

Technical Field

[0001] This invention belongs to the field of kidney pathology detection technology, specifically relating to a method for detecting type IV collagen expression in kidney biopsy tissue. Background Technology

[0002] Collagen type IV (CIV) is a network structure formed by molecular cross-linking. It is a major component of the hepatic basement membrane, found in the portal vein region, around the central vein, and distributed along the sinusoids. During its synthesis and metabolism, CIV does not require the removal of telopeptides and is deposited in the extracellular matrix; therefore, elevated serum CIV levels may reflect an accelerated turnover rate of the hepatic sinusoidal basement membrane. Basic and clinical studies have found a close correlation between serum CIV levels and the degree of liver fibrosis and portal hypertension, but a smaller correlation with liver inflammatory activity. Therefore, measuring serum CIV collagen content is an important indicator for the auxiliary diagnosis of liver fibrosis. Besides its auxiliary diagnostic role in liver fibrosis, serum CIV is a useful indicator for assessing early diabetic kidney damage or the degree of glomerular fibrosis. Most CIV collagen in the glomerular basement membrane is formed by the interweaving of α3, α4, and α5 chains.

[0003] Alport syndrome is a hereditary kidney disease caused by mutations in the genes encoding type IV collagen α3 / α4 / α5 chains. Clinically, it manifests as hematuria, proteinuria, and progressive renal failure. Some patients also experience deafness and ocular changes. Patients primarily present with persistent glomerular hematuria or hematuria accompanied by proteinuria can be diagnosed with Alport syndrome by detecting abnormal immunofluorescence staining of type IV collagen α3, α4, and α5 chains in the glomerular basement membrane (GBM). Therefore, detecting the fluorescence staining of type IV collagen α3, α4, and α5 chains provides important evidence for the diagnosis of Alport syndrome.

[0004] Immunofluorescence staining of paraffin-embedded tissues continues to play a vital role in basic research and clinical pathological diagnosis. It is particularly important in the localization of multiple markers, identification of membrane antigens, identification of antibody types (fluorescently labeled antibodies), localization of trace proteins, and pathological diagnosis of renal biopsies. However, after formalin fixation, calcium... 2+ The tight complexes formed by other divalent ions and proteins block the antigen, hindering antigen-antibody binding during immunofluorescence staining on paraffin sections. This directly affects the specificity and sensitivity of the staining results, consequently impacting the accuracy and detection rate of the detection. Therefore, paraffin sections are almost never used as a detection material for type IV collagen expression detection.

[0005] Currently, the routine method for staining type IV collagen is to use frozen tissue sections for immunofluorescence staining to detect the expression of type IV collagen. However, due to factors such as sampling and segmentation, not all renal biopsy tissues contain glomeruli. Therefore, it is impossible to observe the expression of type IV collagen in frozen sections without glomeruli. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a method for detecting type IV collagen expression in renal biopsy tissue, which can improve the accuracy and detection rate of type IV collagen expression in renal biopsy tissue.

[0007] The following technical solutions are used to achieve the above objectives.

[0008] The first aspect of this invention provides a method for detecting type IV collagen expression in renal biopsy tissue, comprising the following steps:

[0009] The paraffin sections of renal biopsy tissue were dewaxed and hydrated to obtain the processed tissue sections.

[0010] The treated tissue sections were added to the repair solution, heated to boiling, and then cooled to room temperature to obtain the repaired tissue sections.

[0011] The repaired tissue sections were added to protease for enzymatic digestion to obtain enzymatically digested tissue sections.

[0012] The primary antibody was added to the tissue sections after enzymatic digestion for initial incubation, followed by incubation with a fluorescein-labeled secondary antibody against the primary antibody.

[0013] In some embodiments, the heating and boiling time is 4.5 min to 5.5 min; preferably, the heating and boiling time is 4.8 min to 5.2 min.

[0014] In some embodiments, the enzymatic hydrolysis time is 2 min to 3 min, and the concentration of the protease is 120 ug / ml to 130 ug / ml; preferably, the enzymatic hydrolysis time is 2.5 min to 3 min, and the concentration of the protease is 123 ug / ml to 127 ug / ml.

[0015] In some embodiments, the protease is pepsin.

[0016] In some embodiments, the remedy is an EDTA buffer solution with a pH of 8 to 9.

[0017] In some embodiments, the fluorescein is fluorescein isothiocyanate.

[0018] In some embodiments, the following steps are included before the repair solution is added to the processed tissue sections:

[0019] Preheat the repair solution to 90℃~99℃.

[0020] In some embodiments, the thickness of the paraffin sections of the renal biopsy tissue is 1.3 μm to 1.7 μm.

[0021] In some embodiments, the step of adding the repaired tissue slices to a protease for enzymatic digestion includes:

[0022] The repaired tissue sections were washed with phosphate buffer.

[0023] After cleaning, the moist tissue slices were added to protease for enzymatic hydrolysis.

[0024] After enzymatic digestion, the tissue sections were washed with phosphate buffer.

[0025] In some embodiments, prior to washing the repaired tissue section with phosphate buffer, an immunohistograph is used to draw a closed loop around the repaired tissue section, the closed loop enclosing the tissue section.

[0026] In this invention, the inventors selected paraffin sections of renal biopsy tissue as the detection carrier. During the detection of type IV collagen expression in renal biopsy tissue using immunofluorescence technology, the paraffin sections were dewaxed and then subjected to high-temperature repair combined with protease digestion. Through appropriate processing and strict process control, it was found that the α3, α4, and α5 chains of type IV collagen could be fully displayed. This allowed the α3, α4, and α5 chains of type IV collagen to fully bind with the antibody during antibody fluorescence detection, improving the specificity, accuracy, and sensitivity of the stained structure. This method can accurately detect type IV collagen expression in renal biopsy tissue with or without glomeruli, effectively increasing the detection rate of type IV collagen in renal biopsy immunofluorescence. Furthermore, by optimizing the duration of high-temperature repair, the duration of protease digestion, and the concentration of the protease, the fluorescence expression effect of type IV collagen could be further improved. Therefore, this method for detecting type IV collagen expression in renal biopsy tissue is suitable for detecting type IV collagen expression in renal biopsy tissue where there are no glomeruli or too few glomeruli. It has high specificity and can provide a new detection method for Alport syndrome in cases where there are no glomeruli in renal biopsy tissue. Attached Figure Description

[0027] Figure 1 This is an immunofluorescence image of paraffin sections of kidney biopsy tissue without glomeruli from a normal population in Example 1.

[0028] Figure 2 This is an immunofluorescence image of paraffin sections of renal biopsy tissue without glomeruli from an Alport syndrome case in Example 1.

[0029] Figure 3 This is an immunofluorescence result image of frozen sections of renal biopsy tissue without glomeruli from normal individuals in Example 2.

[0030] Figure 4 This is an immunofluorescence image of frozen sections of glomerular tissue from renal biopsies of normal individuals in Example 2.

[0031] Figure 5 This is an immunofluorescence image of frozen sections of glomerular tissue from a kidney biopsy in Example 2 of an Alport syndrome case. Detailed Implementation

[0032] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0033] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0034] Unless otherwise defined, all technical and scientific terms used in this invention are consistent with those belonging to this invention.

[0035] The meanings are generally understood to be the same by those skilled in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0036] The following are specific embodiments.

[0037] Example 1

[0038] This embodiment provides a method for detecting type IV collagen expression in renal biopsy tissue, comprising the following steps:

[0039] 1. Pretreatment of tissues: Paraffin sections of renal biopsy tissue without glomeruli from Alport syndrome cases were used as experimental group 1, and paraffin sections of renal biopsy tissue without glomeruli from normal individuals were used as experimental group 2. The thickness of the paraffin sections was 1.5 micrometers, and they were dewaxed to water.

[0040] The dewaxing process involves immersing the paraffin slices in water according to the following steps to perform the dewaxing operation:

[0041] (1) Xylene (I) 10 min

[0042] (2) Xylene (II) 10 min

[0043] (3) Xylene: (III) 10 min

[0044] (4) 100% ethanol (Ⅰ) 5 min -- 100% ethanol (Ⅱ) 5 min -- 80% ethanol 5 min -- distilled water 5 min.

[0045] The above treatment yielded dewaxed tissue sections.

[0046] 2. Antigen retrieval: Place 1L of 1:50 pH 8.0 EDTA buffer in a pot and heat for about 7 minutes to preheat the EDTA buffer to 99°C. Then immerse the tissue sections in the EDTA buffer and continue heating to boiling for 5 minutes. Place the pot in cold water to cool and allow the retrieval solution to return to room temperature.

[0047] 3. Circle drawing: Remove the tissue section, wipe the liquid around the tissue section dry, and use an immunohistograph pen (DAKO) to draw a circle around the tissue section to form a closed loop, preventing antibodies from escaping the circle and causing false negatives or false positives.

[0048] 4. Protease digestion: Wash the tissue sections 3 times for 3 minutes each with PBS solution (PBS solution is prepared by mixing 4.6g Na2HPO4, 0.26g NaH2PO4, 8.5g NaCl, 1ml Tween, 3ml 1mol hydrochloric acid to a pH of 7.2-7.4, and 1L distilled water). Remove excess PBS solution from the tissue sections (keep them moist, but avoid drying them out, but also avoid leaving too much PBS solution to prevent antibody dilution). Place the tissue sections in an incubator and add 125ug / ml pepsin for digestion. Digest at room temperature for 3 minutes, then wash with PBS 3 times for 3 minutes each.

[0049] 5. Add mouse anti-human type IV collagen primary antibody (Jiangsu Lanou) of α1 chain, α3 chain and α5 chain, and rabbit anti-human type IV collagen primary antibody (Abnova) of α4 chain, then incubate in a constant temperature incubator at 37℃ for 35 min, followed by washing with PBS 3 times x 3 min.

[0050] Add fluorescein isothiocyanate-labeled mouse anti-type IV collagen secondary antibody (DAKO Anti-Mouse immunoglobulins / FITC) and rabbit anti-type IV collagen secondary antibody (DAKO Anti-Rabbit immunoglobulins / FITC), then incubate at 37°C for 40 min, and wash 3 times with PBS for 3 min each time.

[0051] 6. Gently wipe the liquid around the tissue section dry, add glycerin, cover with a coverslip, and observe and acquire images under a microscope.

[0052] 7. Results:

[0053] Figure 1 Images (a)-(d) are immunofluorescence results of paraffin sections of kidney biopsies from experimental group 1, showing tissue without glomeruli. Figure 1 (a) The α1 chain positive control is normal. Figure 1 (b) The α3 chain is shown to be normally expressed in the glomerular basement membrane and renal tubular basement membrane; Figure 1 (c) shows that the α4 chain is normally expressed in the glomerular basement membrane and renal tubular basement membrane; Figure 1 (d) shows that the α5 chain is normally expressed in the glomerular basement membrane, Burman's capsule, and renal tubular basement membrane.

[0054] Figure 2 (a)-(d) are immunofluorescence results of paraffin sections of renal biopsies from Alport syndrome cases in experimental group 2, showing no glomerular tissue. Figure 2 (a) The α1 chain positive control is normal. Figure 2 (b) Shows loss of α3 chain expression in the glomerular basement membrane and renal tubular basement membrane; Figure 2 (c) shows that the α4 chain is absent in the glomerular basement membrane and renal tubular basement membrane; Figure 2 (d) shows the absence of α5 chain expression in the glomerular basement membrane, Burman's capsule, and renal tubular basement membrane.

[0055] Example 2

[0056] The method for detecting type IV collagen expression in renal biopsy tissue provided in this embodiment includes the following steps:

[0057] 1. Tissue pretreatment: Fluorescent renal biopsy specimens were collected from normal individuals and Alport syndrome cases, respectively. The specimens were subjected to routine in vitro fixation, washing and fixative solution, frozen sectioning (thickness of 3 micrometers), and acetone fixation to obtain three groups of sections: control group (freezing renal biopsy tissue of normal individuals without glomeruli), experimental group 3 (freezing renal biopsy tissue of normal individuals with glomeruli), and experimental group 4 (freezing renal biopsy tissue of Alport syndrome cases with glomeruli).

[0058] 2. Pretreatment solution treatment: Circle the slides to form α1, α3, a4 and a5 regions. Treat the α1 and α3 regions with antigen retrieval solution (Jiangsu Lanou) and incubate at room temperature for 15 min. Add PBS solution to the a4 and a5 regions to prevent the slides from drying out.

[0059] 3. Add mouse anti-human type IV collagen primary antibody (Jiangsu Lanou) with α1 chain, α3 chain and α5 chain, and rabbit anti-human type IV collagen primary antibody (Abnova) with α4 chain, and incubate in a constant temperature incubator at 37℃ for 35 min, followed by washing with PBS 3 times x 3 min.

[0060] 4. Add fluorescein isothiocyanate-labeled secondary antibody (DAKO Anti-Mouse immunoglobulins / FITC) and rabbit anti-type IV collagen secondary antibody (DAKO Anti-Rabbit immunoglobulins / FITC), then incubate at 37°C for 40 min, and wash 3 times with PBS for 3 min each time.

[0061] 5. Gently wipe the liquid around the tissue section dry, add glycerin, cover with a coverslip, and observe and acquire images under a microscope.

[0062] 6. Results:

[0063] Figure 3 Images (a)-(d) are immunofluorescence results of frozen sections of renal biopsies from healthy individuals without glomeruli. Figure 3 As shown in (a)-(d), the expression of glomeruli and type IV collagen could not be observed in the immunofluorescence results of frozen sections of renal biopsy tissue without glomeruli.

[0064] Figure 4 Images (a)-(d) are immunofluorescence results of frozen sections of glomerular tissue from renal biopsies in normal individuals. Figure 4 (a) The α1 chain positive control is normal. Figure 4 (b) The α3 chain is shown to be normally expressed in the glomerular basement membrane and renal tubular basement membrane; Figure 4 (c) shows that the α4 chain is normally expressed in the glomerular basement membrane and renal tubular basement membrane; Figure 4 (d) shows that the α5 chain is normally expressed in the glomerular basement membrane, Burman's capsule, and renal tubular basement membrane.

[0065] Figure 5 Images (a)-(d) are immunofluorescence results of frozen sections of glomerular tissue from renal biopsies in Alport syndrome cases. Figure 5 (a) The α1 chain positive control is normal. Figure 5 (b) Shows loss of α3 chain expression in the glomerular basement membrane and renal tubular basement membrane; Figure 5 (c) shows that the α4 chain is absent in the glomerular basement membrane and renal tubular basement membrane; Figure 5 (d) shows the absence of α5 chain expression in the glomerular basement membrane, Burman's capsule, and renal tubular basement membrane.

[0066] The expression of type IV collagen in Examples 1 and 2 above is summarized in Table 1 below:

[0067]

[0068] Table 1 shows that, in experimental groups 3-4, the expression of α3, α4, and α5 chains of type IV collagen could be detected normally in frozen sections of renal biopsy tissue with normal glomeruli, thus enabling the diagnosis of Alport syndrome. However, in the control group, the expression of type IV collagen could not be observed via immunofluorescence in frozen sections of renal biopsy tissue without normal glomeruli, thus failing to provide any diagnostic value for Alport syndrome. Therefore, it is evident that the expression of type IV collagen via immunofluorescence cannot be observed in frozen sections of renal biopsy tissue without glomeruli. In experimental groups 1-2, by dewaxing paraffin-embedded type IV collagen tissue from renal biopsy tissue, followed by high-temperature repair with a repair solution, and then digestion with pepsin followed by antibody fluorescence detection, the expression of α3, α4, and α5 chains of type IV collagen could still be accurately detected in renal biopsy tissue without glomeruli. This provides a new detection method for Alport syndrome in renal biopsy tissue without glomeruli.

[0069] Furthermore, the analysis of the glomerulonephrosis rate in frozen tissue and the addition rate of type IV collagen in non-recent specimens showed that, in the absence of glomeruli in renal biopsy tissue, the detection method of type IV collagen expression by detecting type IV collagen expression in renal biopsy paraffin sections could improve the detection rate of type IV collagen by 10% compared to the fluorescence immunoassay method using frozen sections of renal biopsy tissue.

[0070] Example 3

[0071] In this embodiment, during the antigen retrieval step, paraffin sections of renal biopsy tissue without glomeruli from normal individuals were boiled in EDTA buffer for 5 min, 10 min, and 15 min, respectively. During the protease digestion step, the tissue sections were digested with pepsin for 3 min, 5 min, and 10 min, with pepsin concentrations of 125 ug / ml and 250 ug / ml. The experiments were conducted using the above variables, with each experiment repeated 5 times. Other relevant reaction conditions were performed according to Example 1. The fluorescence effect of type IV collagen expression is shown in Table 2 below.

[0072] The method for determining the fluorescence effect of type IV collagen expression is as follows:

[0073] Poor expression: no fluorescence or only appears visible under high magnification.

[0074] The effect is average: it appears to be visible under low magnification, but is clearly visible under high magnification.

[0075] The expression effect is good: it is clearly visible under low magnification and clearly visible under high magnification.

[0076] Excellent expressive effect: clearly visible under low magnification and dazzling under high magnification.

[0077] Table 2. Effects of heating time, enzymatic hydrolysis time, and pepsin concentration on the fluorescence effect of type IV collagen expression.

[0078]

[0079] Based on the results in Table 2, the protease concentration was further selected as 125 ug / ml. Paraffin sections of renal biopsy tissue without glomeruli from normal individuals were added to EDTA buffer and boiled for 5 min and 10 min, respectively. In the protease digestion step, the tissue sections were digested by pepsin for 1 min, 2 min, and 2.5 min. The experiment was set up with the above variables, and other related reaction conditions were carried out according to Example 1. The fluorescence effect of type IV collagen expression is shown in Table 3 below.

[0080] Table 3. Effects of heating time, enzymatic hydrolysis time, and pepsin concentration on the fluorescence effect of type IV collagen expression.

[0081] Enzymatic hydrolysis at 125ug / ml for 1 min Enzymatic hydrolysis at 125ug / ml for 2 minutes Enzymatic hydrolysis at 125ug / ml for 2.5min Heating time: 5 minutes Poor expression effect Good expression effect Good expression effect Heating time: 10 minutes Poor expression effect The expressive effect was average. The expressive effect was average.

[0082] Based on the results in Table 2, the protease concentration was further set to 125 ug / ml and 200 ug / ml. Paraffin sections of renal biopsy tissue without glomeruli from normal individuals were added to EDTA buffer and boiled for 4 min, 4.5 min, and 5.5 min, respectively. In the protease digestion step, the tissue sections were digested by pepsin for 3 min. The experiment was conducted with the above variables, and other related reaction conditions were carried out according to Example 1. The fluorescence effect of type IV collagen expression is shown in Table 4 below.

[0083] Table 4. Effects of heating time, enzymatic hydrolysis time, and pepsin concentration on the fluorescence effect of type IV collagen expression.

[0084] Enzymatic hydrolysis at 125ug / ml for 3 minutes Enzymatic hydrolysis at 200ug / ml for 3 minutes Heating time: 4 minutes The expressive effect was average. The expressive effect is average. Heating time: 4.5 min Good expression effect The expressive effect was average. Heating time: 5.5 min Good expression effect The expressive effect was average.

[0085] Table 2 shows that heating / boiling time, enzymatic hydrolysis time, and enzyme concentration all affect the fluorescence expression of type IV collagen. Tables 3 and 4 show that these three factors interact to influence the fluorescence expression of type IV collagen. Our experiments revealed that enzyme concentration, hydrolysis time, and boiling time significantly impact the experimental results. High or low enzyme concentration and short hydrolysis time can lead to poor or excessive hydrolysis. Boiling time affects the opening of antigenic determinants and the tissue background of the slide. Therefore, controlling the hydrolysis time to 2-3 min, the protease concentration to 123-127 ug / ml, and the heating / boiling time to 4.5-5.5 min resulted in better fluorescence expression of type IV collagen. Observation showed that the optimal combination of heating / boiling time (5 min), hydrolysis time (3 min), and pepsin concentration (125 ug / ml) for type IV collagen immunofluorescence expression was achieved.

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting type IV collagen expression in renal biopsy tissue, characterized in that, Includes the following steps: The paraffin sections of renal biopsy tissue were dewaxed and hydrated to obtain the processed tissue sections. The treated tissue sections were added to the repair solution, heated to boiling, and then cooled to room temperature to obtain the repaired tissue sections. The heating and boiling time is 4.5 min to 5.5 min; The repaired tissue sections were added to a protease for enzymatic hydrolysis to obtain enzymatically hydrolyzed tissue sections; the hydrolysis time was 2 min to 3 min, and the concentration of the protease was 120 ug / ml to 130 ug / ml; the protease was pepsin. The tissue sections after enzymatic digestion were initially incubated with primary antibody, followed by incubation with fluorescein-labeled secondary antibody against the primary antibody. The repair solution is an EDTA buffer solution, and the pH value of the EDTA buffer solution is 8-9; Before adding the repair solution to the processed tissue sections, the following steps are also included: Preheat the repair solution to 90°C~99°C; The thickness of the paraffin sections of the renal biopsy tissue was 1.3 μm to 1.7 μm; The steps involved in adding protease to the repaired tissue sections for enzymatic digestion include: The repaired tissue sections were washed with phosphate buffer. After cleaning, the moist tissue slices were added to protease for enzymatic hydrolysis. After enzymatic digestion, the tissue sections were washed with phosphate buffer. Before washing the repaired tissue section with phosphate buffer, the procedure includes drawing a closed loop around the repaired tissue section using an immunohistograph pen, the closed loop enclosing the tissue section.

2. The detection method as described in claim 1, characterized in that, The heating and boiling time is 4.8 min to 5.2 min.

3. The detection method as described in claim 1, characterized in that, The enzymatic hydrolysis time is 2.5 min to 3 min, and the concentration of protease is 123 ug / ml to 127 ug / ml.

4. The detection method according to any one of claims 1 to 3, characterized in that, The fluorescein is fluorescein isothiocyanate.

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