参见作者原研究论文

本实验方案简略版
Dec 2015
Advertisement

本文章节


 

CD45 Immunohistochemistry in Mouse Kidney
小鼠肾脏中的 CD45 免疫组织化学   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

CD45 is a pan-leukocyte marker, and CD45 stain is widely used to determine the extent of inflammatory cell infiltration and its association with tissue injury. In this manuscript, we share a reliable immunohistochemistry (IHC) protocol for CD45 staining in sections of paraffin-embedded mouse kidney. A rat anti-CD45 antibody was used as primary antibody, and a mouse adsorbed biotin-conjugated goat anti-rat IgG was selected as secondary antibody. A horseradish peroxidase (HRP)-linked avidin/biotin detection system was used to amplify the signal, which was detected with 3,3′-Diaminobenzidine (DAB). With this protocol, we show that the CD45 antibody recognizes cells of hematolymphoid lineage in bone marrow, as well as monocyte/macrophages in liver and lung tissue. The utility of this protocol in pathology research was indicated by dramatically increased CD45-positive (CD45+) cells in the kidneys of a mouse model of diabetes. Double staining for CD45 and injury marker KIM-1 showed accumulated CD45+ cells around injured tubular cells. CD45 and F4/80 macrophage staining on adjacent tissue sections revealed overlap of CD45+ cells with other inflammatory cells.

Keywords: CD45 (CD45), Diabetes (糖尿病), Kidney (肾脏), KIM-1 (KIM-1), IHC (免疫组化), OVE (OVE)

Background

CD45 is a pan-leukocyte protein with tyrosine phosphatase activity involved in the regulation of signal transduction in hematopoiesis. It belongs to the receptor type protein of tyrosine phosphatase family. It is heavily glycosylated and expressed at high levels on nucleated hematopoietic cells, including granulocytes, lymphocytes, macrophages/histiocytes, mast cells, monocytes, basophils, and plasma cells.


Clinically, CD45 is mainly used to confirm the presence of inflammatory cells and the hematopoietic nature of tumors. In basic research, CD45 is widely used as a pan-leukocyte marker in flow cytometry and histology studies.


Since the method for IHC detection of CD45 in mouse tissue is of broad interest to the scientific community, we therefore share our optimized protocol (Zheng et al., 2016) in detail in this manuscript. We present images from tissues with known CD45 positive (CD45+) cells and focus on kidneys of diabetic mice, where we demonstrate dramatically increased diabetes-induced CD45 expression, which co-localized with other biomarkers in the kidney.

Materials and Reagents

  1. VWR micro cover glass (VWR, catalog number: 48393-081)

  2. Microslides (Superfrost Plus, catalog number: 48311-703, VWR, USA)

  3. ImmEdge pen (Vector, catalog number: H-4000)

  4. Primary antibodies:

    1. CD45 (Angio-Proteomie, catalog number: mAP-0058; Species: rat; Target: mouse; Dilution: 1:400)

    2. Kim-1 (R&D Systems, catalog number: AF1817; Species: goat; Target: mouse; Dilution: 1:500)

    3. F4/80 (Serotec, catalog number: MCA497R, Species: rat; Target: mouse; Dilution: 1:100)

  5. Secondary antibodies:

    1. Anti-rat, mouse adsorbed (Vector, catalog number: BA-9401; Species: goat; Target: rat; Dilution: 1:200; Comjugated: Biotinylated)

    2. Anti-goat (Jackson Labs, catalog number: 705-095-147; Species: donkey; Target: goat; Dilution: 1:100; Comjugated: FITC)

    3. Anti-rat (Jackson Labs, catalog number: 712-095-150; Species: donkey; Target: rat; Dilution: 1:100; Comjugated: Cy3)

  6. Target Retrieval solution (10× concentrated; Dako, catalog number: S1699)

  7. 30% H2O2 (Sigma-Aldrich, catalog number: 216763-500 ml)

  8. Avidin/Biotin Blocking Kit (Vector Laboratories, catalog number: SP-2001, ZF0917)

  9. Vectastin Elite ABC Kit (Peroxidase (Standard), catalog number: PK-6100, ZF1011)

  10. DAB substrate kit (50× DAB Chromogen; ABCAM, catalog number: ab64238)

  11. Goat serum (Jackson ImmunoResearch, catalog number: 005-000-121)

  12. Donkey serum (Jackson Immunoresearch, catalog number: 017000121)

  13. Hematoxylin solution (Merck, catalog number: 1.05175.0500)

  14. Formalin (10% Buffered Formalin; Fisher Scientific, catalog number: 23-427098)

  15. Reagents Xylenes (Purified, catalog number: UX-78923-75, Cole-Parmer)

  16. VectaMount (permanent Mounting medium; Vector, catalog number: H-5000)

  17. VECTASHIELD HardSet (Antifade mounting medium with DAPI; Vector Laboratories, catalog number: H-1500)

  18. Sodium citrate tribasic dihydrate (Sigma, catalog number: S4641)

  19. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na2) (Sigma, catalog number: E5134)

  20. Sodium hydroxide pellets (EMD, catalog number: SX0593)

  21. Citrate buffer (see Recipes)

  22. 14% EDTA (see Recipes)

  23. 0.1% Tween 20 in PBS (see Recipes)

  24. 2.5% normal goat serum (see Recipes)

  25. 2.5% normal donkey serum (see Recipes)

  26. PBS-T (see Recipes)

Equipment

  1. Microscopes (Nikon, ECLIPSE, E600 and ZEISS, AXIO, IMAGER.A1 Fluorescent microscope)

  2. Incubation box: StainTray 20 Place with Black Lid, Newcomer Supply Inc. # 6847-20BL (https://www.newcomersupply.com/product/staintray-20-place/)

  3. Staining jar (Coplin Staining Jar with Cover, Wheaton; VWR, catalog number: 25460-000)

  4. Hotplates (Stirrers and Hotplate Stirrers, Benchmark Scientific, catalog number: H-4000-HS)

Procedure

  1. Tissue slides

    The slides used were obtained from mouse tissue, specifically bone marrow, lung, liver, and kidney. All tissues were processed by standard formalin fixation, paraffin embedding, and tissue block sectioning, except for bone marrow of mouse tibia, which was decalcified between formalin fixation and paraffin embedding. For this purpose, the tibia bone was isolated from normal adult C57/Bl6 mice, fixed with formalin for 48 h, decalcified in 14% EDTA solution for 72 h before embedding with paraffin. Kidneys were harvested from the diabetic OVE26 mice [a transgenic mouse strain with early and persistent hyperglycemia (Epstein et al., 1989), and identified as a reliable mouse model of diabetic nephropathy (Zheng et al., 2004)] and uninephrectomized OVE26 mice (Zheng et al., 2011). Non-diabetic control kidney was obtained from age matched, same strain FVB mice. These animals have been tested for albuminuria, and their kidneys have been histologically evaluated with standard hematoxylin and eosin (H & E) and Masson’s trichrome stain. The kidneys with obvious tubulointerstitial fibrosis were used for demonstration of CD45 staining. The tissue sections of kidney, liver, and lung were cut at 4 µm, and sections of tibia were cut at 5 µm. Sections were mounted on positively charged slides. Prior to staining, all paraffin sections were heated for 30 min at 70°C. Bone marrow tissue was considered a positive control, and sections with omitted CD45 primary antibody were used as negative control.


  2. CD45 staining

    Immunohistochemistry (IHC)

    1. Deparaffinize and rehydrate tissue: dewax tissue slides in xylene and rehydrate with graded ethanol to water. (In detail: three changes in xylenes, 5 min each; two changes in 100% ETOH, 3 min each; followed by 95% ETOH, 90% ETOH, and 80% ETOH, 3 min each. Then, move slides to water, 3 min each with two changes.)

    2. Block internal peroxidase activity with H2O2: soak slides in the Coplin jar with 3% H2O2/PBS, 20 min, at room temperature (RT).

    3. Wash the slides with PBS: soak slides in PBS for 5 min, with three changes.

    4. Retrieve antigen in buffer of choice. For this protocol, antigen retrieval was performed in citrate buffer at pH 6, 95-100°C, 20 min. [In detail: Add 50 ml of 1× citrate buffer to a glass Coplin jar, and place this jar in a water bath beaker on top of a heating plate. As soon as the water bath is boiling, insert the tissue slides into the Coplin jar (maximal slide number: 10). Stop heating 20 min later by removing the beaker from the heating plate. Cool the Coplin jar at RT for 30 min.]

    5. Wash the slides with PBS for 5 min three times.

    6. Draw an hydrophobic circle around the tissue section on the slide: circle the tissue section with ImmEdge® Hydrophobic Barrier PAP Pen for a heat-stable, water-repellent barrier to keep reagents localized on tissue specimens and prevent mixing of reagents between sections.

    7. Block the tissue section with normal serum at RT for 30 to 60 min (avidin/biotin blocking procedure was added in this step and step 10).

      1. Prepare 2.5% normal goat serum in PBS.

      2. Add avidin solution from Avidin/Biotin blocking kit to 2.5% normal goat serum (4 drops per 1 ml), mix well.

      3. Place the slides in an incubation box.

      4. Apply 50-100 µl of blocking serum (with Avidin solution) to each tissue section.

      5. Incubate the section at RT for 30 to 60 min.

    8. Wash the slides with PBS-T, 5 min three times.

    9. Apply primary antibody**:

      1. Prepare antibody diluent: add biotin blocking solution to 2.5% goat serum/PBS (4 drops per 1 ml).

      2. Dilute CD45 antibody 1:400 with prepared diluent.

      3. Apply diluted antibody to tissue section.

      4. Incubate the slides in the incubation box overnight at 4°C.

    10. Wash the slides with PBS-T for 5 min three times.

    11. Apply secondary antibody:

      1. Prepare the antibody by diluting the biotinylated goat anti-rat (mouse absorbed) IgG in PBS at 1:200-1:400 dilution.

      2. Apply to tissue sections and incubate the slides in the wet box for 60 min at RT.

    12. Wash with PBS-T 5 min five times.

    13. Apply ABC solution***:

      1. Prepare ABC reagent from VECTASTAIN Elite ABC kit: add two drops (100 μl) of Reagent A to 5 ml of PBS and mix; then add two drops (100 μl) of Reagent B to the above tube and mix immediately.

      2. Apply 50-100 µl of ABC reagent to each tissue section, incubate for 30 min at RT.

    14. Wash with PBS-T 5 min three times.

    15. Apply DAB:

      1. Prepare DAB reagent by mixing 200 µl of 50× DAB Chromogen with 10 ml of DAB substrate buffer.

      2. Apply DAB to tissue sections for a maximum of 10 min.

    16. Wash the slides in water 5 min three times.

    17. Counterstain nuclei with hematoxylin, if desired.

    18. Wash the slides with water 5 min three times.

    19. Dehydrate with ethanol and then xylene. (In detail: one incubation in 90% ETOH and two in 100% ethanol, 1 min each, followed by three incubations in xylene, 3 min each.)

    20. Apply permanent mounting medium to the slides and cover with cover glass.

    21. Observe the slides under the microscope to check if the brown color from DAB developed in the positive control and if the negative control section (where the primary antibody was omitted) has no color.

    22. If the staining is good, check the target sections for:

      1. Presence of positive staining.

      2. Location of the positive staining (parenchyma or interstitial tissue).

      3. Record the pathological changes of the tissue section imaged at the selected magnification. If necessary, perform quantitative analysis. Keep the observer blind to the group identification.

    Notes:

    1. **For co-localized staining of CD45 with F4/80, select a tissue slide with three identically aligned serial sections. CD45 and F4/80 antibody are applied separately to adjacent sections. The third section lacks any primary antibody. All the other steps are the same.

    2. ***Allow ABC Reagent to stand for 15-30 min before use.


    Immunofluorescence (IF): Double staining for CD45 and KIM-1

    1. Deparaffinize and rehydrate tissue: tissue slides are dewaxed in xylene and rehydrated with graded ethanol to water.

    2. Retrieve antigen in buffer of choice. For this protocol, antigen retrieval was performed in citrate buffer at pH 6, 95-100°C, 20 min. (In detail: Add 50 ml of 1× citrate buffer to a glass Coplin jar, and place this jar in a water bath beaker on top of a heating plate. As soon as the water bath is boiling, insert the tissue slides into the Coplin jar (maximal slide number: 10). Stop heating 20 min later by removing the beaker from the heating plate. Cool the Coplin jar at RT for 30 min.)

    3. Wash the slides with PBS for 5 min.

    4. Draw a hydrophobic circle around the tissue section on the slide: circle the tissue section with ImmEdge® Hydrophobic Barrier PAP Pen for a heat-stable, water-repellent barrier to keep reagents localized on tissue specimens and prevent mixing of reagents between sections.

    5. Serum blocking:

      1. Prepare 2.5% normal donkey serum in PBS.

      2. Apply 50-100 µl of blocking serum to cover the tissue section.

      3. Incubate the slides for 60 min at RT.

    6. Rinse the slides briefly with PBS-T for 3 min three times.

    7. Apply primary antibody:

      1. Prepare the mixed antibody solution: add 5 µl of CD45 antibody (1:400) and 4 µl of KIM-1 antibody (1:500) into 2 ml of 2.5% donkey serum/PBS.

      2. Apply this mixed antibody solution to the designated tissue sections.

      3. Apply pre-immune serum only to the section which will be used as negative control.

      4. Incubate the slides overnight at 4°C.

    8. Wash the slides with PBS-T 5 min three times.

    9. Apply secondary antibody:

      1. Prepare antibody mix by adding Cy3 conjugated anti-Rat IgG and FITC conjugated anti-goat IgG (both at 1:100 dilution) to PBS and mix well.

      2. Apply this mixed antibody to tissue section.

      3. Incubate the slides for 60 min in the dark at RT.

    10. Wash the slides in the dark with PBS-T for 5 min five times.

    11. Mount the tissue sections using mounting medium with DAPI, overlay a cover glass, and store the slides in the dark at 4°C.

    12. Check the slides using a fluorescence microscope to validate positive and negative staining. In this study, DAPI/FITC/TRITC filters were used to reveal nuclei (DAPI), KIM-1 (FITC), and CD45 (Cy3) staining.

    13. Once the IF method is validated, check the target sections for positive stain and location. At the selected magnification, capture the images of the sample sequentially by DAPI, FITC, and TRITC filters with multi-channel acquisition settings and merge immunofluorescence images using the software. Keep the background and exposure equal for image comparison. If necessary, perform quantitative analysis. Keep the observer blind to the group identification.

    Data analysis

    1. Validate the CD45 antibody in mouse tissue.

      Successful IHC staining requires adequate antibody sensitivity and specificity. CD45 is a transmembrane cell surface glycoprotein expressed by the vast majority of cells of hematolymphoid lineage. Therefore, bone marrow seemed a good positive control tissue. Bone marrow requires a decalcification process for sectioning. In this study, bone marrow of mouse tibia was decalcified with 14% EDTA for 72 h. The results (Figure 1) show that CD45 antigenicity was retained after decalcification. As shown in Figure 1A, many nucleated cells in bone marrow stained positive for CD45. The CD45+ cells (solid arrow) were especially obvious when the nucleated cells resided within the red blood cell (RBC) islands (Figure 1C). The positive stains were contrasted with negatively stained cells, such as megakaryocytes (the precursor for platelets), adipocytes, and the surrounding bone tissue. When the CD45 antibody was omitted, staining was negative (Figure 1B). These results show that the optimized protocol produced sensitive and specific staining with this CD45 antibody.



      Figure 1. Representative CD45 staining in normal adult mouse tissue sections from bone marrow, lung, and liver.

      Normal bone marrow contained many CD45+ cells (A), which were absent if the CD45 antibody was omitted (B). At higher magnification (C), the population of CD45 positive bone marrow cells (solid arrow) did not appear to include RBCs, megakaryocytes (open arrow), adipocytes, or surrounding bone tissue. In normal lung (D), and liver (E), CD45 was expressed in a small number of widely distributed cells, likely macrophages in the inter-alveolar septum of the lung and Kupffer cells in the sinusoids of the liver. Staining was performed in formalin-fixed paraffin-embedded tissue sections, cut at a thickness of 5 µm for bone and 4 µm for lung and liver. Images were taken at 20× or 40× magnification.


      When tissue sections from liver or lung of normal mice were stained, we observed positive cells individually dispersed in the tissue. In addition to the leukocytes in the blood vessels, the distinct CD45+ cells resided in the alveolar septum of lung or in the sinusoids of the liver. These positive cells were most likely the alveolar macrophages in lung or Kupffer cells in liver. Similar CD45 staining in human lung and liver has been well illustrated by a mouse anti-human CD45 antibody. https://www.novusbio.com/PDFs3/NBP2-52868.pdf.

        Overall, this antibody protocol was validated to detect CD45+ cells in normal mouse bone marrow, lung, and liver. Based on these results, we proceeded with CD45 staining in normal and diabetic mouse kidneys.


    2. CD45 staining in normal and diabetic mouse kidneys

      The same CD45 staining protocol was applied to kidneys from normal and OVE diabetic mice. As shown in Figure 2, distinct positive staining in cells was revealed in the presence of a low background. As in bone marrow, no staining was seen in kidney sections if the CD45 antibody was omitted (images not displayed). In normal kidneys, CD45+ cells were distributed evenly throughout the renal cortex (A), primarily in the tubulointerstitial space (C). CD45+ cells did not form clusters in normal kidney. OVE diabetes produced severe albuminuria (Zheng et al., 2004 and 2016), which coincided with drastic changes in the prevalence and pattern of CD45 staining. The number of CD45 positive cells was clearly greatly increased in diabetic kidneys (B). Their distribution was also strikingly changed: CD45+ cells tended to surround dilated or collapsed tubules (B and D), where they formed large, tightly packed clusters that appeared to broaden the interstitial space (E and F). These results clearly show that this CD45 staining protocol worked well in mouse kidney and could be used to analyze major changes produced by diabetes.



      Figure 2. Representative images of CD45 +cells in normal and diabetic kidney.
      In normal kidney, CD45+ cells were distributed throughout the renal cortex (A). Higher magnification of normal kidney revealed that CD45+ cells were mainly localized in the tubulointerstitial space in groups of 1 or 2 positive cells (C). In diabetic kidney, the number of CD45+ positive cells greatly increased, and they were not evenly distributed (B). Most CD45+ cells in diabetic kidney were in the tubulointerstitial space surrounding dilated or collapsed tubules (B and D). CD45+ cells were tightly packed in large clusters that appeared to broaden the interstitial space (E and F). Paraffin-embedded formalin-fixed tissue sections were from normal FVB or OVE diabetic mice with overt albuminuria. CD45 staining was performed as described in the Methods section and visualized by DAB color production. Images in A and B were taken with 20× objective and in C, D, E, and F were taken with 100× objective.



      Figure 3. Typical images of OVE diabetic kidney double stained with CD45 and KIM-1 (A, B, C) or stained on serial sections for co-localization of CD45 and macrophage marker F4/80 (D, E, F).
      CD45 and KIM-1 were double stained using the immunofluorescence protocol described in Methods. Primary antibody was visualized with Cy3 (red) for CD45 and FITC (green) for KIM-1. The kidney injury molecule KIM-1 was mostly observed on the apical surface of epithelial cells in dilated renal tubules (A, original magnification 20×, and B & C, original magnification 40×). CD45+ infiltrating cells were present in the tubulointerstitium nearby tubule staining for KIM-1. The staining pattern of CD45 was compared with F4/80 in kidney sections from uninephrectomized diabetic mice by applying antibodies separately to three serial tissue sections on one slide in IHC; CD45 (D), F4/80 (E), and no primary antibody control (F). CD45 and F4/80 stained the same regions of the renal cortex. Original magnification of D, E, and F was 20×.


    3. Comparison of CD45 staining with KIM-1 or F4/80 staining in diabetic kidney

      To expand our understanding of the pathological significance of increased CD45 staining, we compared it with KIM-1 and F4/80 staining (Figure 3). Kidney injury molecule-1 (KIM-1) is an epithelial phosphatidylserine receptor expressed in acute or chronic fibrotic renal disease (Humphreys et al., 2013; Mori et al., 2021). KIM-1 and CD45 were double stained on the same section by the immunofluorescence protocol described in Methods. Primary antibody was visualized with FITC (green) for KIM-1 and Cy3 (red) for CD45. KIM-1 was expressed on the apical surface of epithelial cells in dilated renal tubules (Figure 3A-C). Clusters of CD45+ infiltrating cells were present in the tubulointerstitium close to many tubules stained for KIM-1. The association between KIM-1 and CD45 staining indicates that the leukocytes were attracted by the injured proximal epitheliums, and it further illustrates the correlation between tubular cell damage and renal inflammation in diabetic kidneys.

        F4/80 is a widely used marker for macrophages. Since CD45 and F4/80 antibodies were both from rat, they could not be distinguished in the same section. Therefore, their staining patterns were compared by applying antibodies separately to close serial tissue sections. As shown in Figure 3D and E, their staining patterns were very similar, implying that many CD45+ cells were macrophages. This strategy could be used with other antibodies of interest.

        In this protocol, we focused on describing the procedures for CD45 staining up to the optional procedure of quantitative analysis. While we have previously and successfully performed quantitative analysis after this CD45 staining protocol (Zheng et al., 2016), staining in different groups is frequently so obvious that quantitation is not needed to reach a conclusion.


      Summary

      In summary, we optimized an IHC protocol for CD45 staining in mouse kidney.

        Since the primary antibody was developed from rat, a biotinylated goat anti-rat (mouse adsorbed) secondary antibody was selected for the staining. To reduce the non-specific binding, the “mouse adsorbed” anti-rat IgG (H+L) products were used because mouse tissues may contain endogenous mouse immunoglobulins. The “biotinylated” antibody can ensure the maximum degree of labeling without compromising the specificity or affinity of the antibody.

        Some tissues have endogenous biotin or biotin-binding proteins, lectins, or non-specific binding substances in the section, which may bind avidin, biotinylated horseradish peroxidase, or other Avidin/Biotin System components, resulting in high background when ABC reagents are used. Therefore, we applied avidin-biotin blocking to minimize background stain. We took an alternative procedure for avidin/biotin blocking in this protocol. The avidin and biotin blocking was incorporated into the step of serum blocking and primary antibody incubation. This procedure worked well in keeping low background in the ABC detection system.

        Antigen unmasking procedures are required to achieve optimum IHC staining for CD45, as this antigen could be masked by prolonged formalin-fixation and the paraffin-embedding process. In this protocol, Heat Induced Epitope Retrieval (HIER) with citrate buffer (pH 6.0) was used. Slides treated in citrate buffer for 20 min at 95-100°C produced adequate results. Antigen retrieval solutions from a commercial source or lab-made are both good (recipe below).

        CD45 is a pan-leukocyte protein that can be used to recognize cells of hematopoietic lineages, such as granulocytes, lymphocytes, and macrophages/monocytes. Many of these cells are normally distributed in the bloodstream and some tissues, such as lung or liver, but are especially enriched in lymph nodes or spleen. These are convenient positive control tissues. Bone marrow tissue can also give a reliable positive stain when mouse tibia bone is decalcified with 14% EDTA for 72 h. A positive stain was absent when the primary antibody was omitted. With this protocol, we demonstrated the significantly increased infiltration of CD45+ cells in kidney from diabetic mice. Double stain or staining in adjacent sections with other antibodies could expand our ability to further associate the infiltration of inflammatory cells with other detrimental factors in the progression of renal diseases.

    Recipes

    1. Citrate buffer (10 mM citric acid, pH 6.0)

      Dissolve 2.94 g sodium citrate tribasic dihydrate in 800 ml of Milli-Q water, adjust to pH 6.0, finalize with Milli-Q water to 1,000 ml.

    2. 14% EDTA

      To make 2,000 ml of 14% EDTA, add 250 g of EDTA-2Na into 1,750 ml of Milli-Q water, followed by 25 g of sodium hydroxide pellets. Keep stirring and heat slightly until the EDTA has dissolved. Adjust pH to 7.0 and bring the volume up to 2,000 ml with water.

    3. 2.5% normal goat serum

      Add 250 µl of normal goat serum to 10 ml of PBS.

    4. 0.1% Tween 20 in PBS

      Add 0.1 ml of Tween 20 to 100 ml of PBS.

    5. 2.5% normal donkey serum

      Add 250 µl of normal donkey serum to 10 ml of PBS.

    6. PBS-T

      0.1% Tween 20 in PBS

    Acknowledgments

    This protocol was adapted and expanded from the previous publications (Zheng et al., 2016). We thank JDRF and NIH for grants support. JDRF 3-2005-932 to SZ, JDRF 1-2005-88, and NIDDK DK072032 to PE.

    Competing interests

    Authors have no conflicts of interest or competing interests to disclose.

    Ethics

    All animal procedures followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Louisville Institutional Animal Care and Use Committee.

    References

    1. Epstein, P. N., Overbeek, P. A. and Means, A. R. (1989). Calmodulin-induced early-onset diabetes in transgenic mice. Cell 58(6): 1067-1073.
    2. Humphreys, B. D., Xu, F., Sabbisetti, V., Grgic, I., Movahedi Naini, S., Wang, N., Chen, G., Xiao, S., Patel, D., Henderson, J. M., et al. (2013). Chronic epithelial kidney injury molecule-1 expression causes murine kidney fibrosis. J Clin Invest 123(9): 4023-4035.
    3. Mori, Y., Ajay, A. K., Chang, J. H., Mou, S., Zhao, H., Kishi, S., Li, J., Brooks, C. R., Xiao, S., Woo, H. et al. (2021). KIM-1 mediates fatty acid uptake by renal tubular cells to promote progressive diabetic kidney disease. Cell Metab 33(5): 1042-1061 e1047.
    4. Zheng, S., Coventry, S., Cai, L., Powell, D. W., Jala, V. R., Haribabu, B. and Epstein, P. N. (2016). Renal Protection by Genetic Deletion of the Atypical Chemokine Receptor ACKR2 in Diabetic OVE Mice. J Diabetes Res 2016: 5362506.
    5. Zheng, S., Noonan, W. T., Metreveli, N. S., Coventry, S., Kralik, P. M., Carlson, E. C. and Epstein, P. N. (2004). Development of late-stage diabetic nephropathy in OVE26 diabetic mice. Diabetes 53(12): 3248-3257.
    6. Zheng, S., Huang, Y., Yang, L., Chen, T., Xu, J. and Epstein, P. N. (2011). Uninephrectomy of diabetic OVE26 mice greatly accelerates albuminuria, fibrosis, inflammatory cell infiltration and changes in gene expression. Nephron Exp Nephrol 119(1): e21-32.

简介

[摘要] CD45 是一种泛白细胞标志物,CD45 染色被广泛用于确定炎症细胞浸润的程度及其与组织损伤的关联。在这份手稿中,我们分享了一个可靠的免疫组织化学 (IHC) 协议,用于在石蜡包埋的小鼠肾脏切片中进行 CD45 染色。使用大鼠抗CD45抗体作为一抗,选择小鼠吸附生物素偶联的山羊抗大鼠IgG作为二抗。使用辣根过氧化物酶 (HRP) 连接的亲和素/生物素检测系统放大信号,并用 3,3'-二氨基联苯胺 (DAB) 检测。通过该协议,我们表明 CD45 抗体可识别骨髓中的血淋巴谱系细胞,以及肝和肺组织中的单核细胞/巨噬细胞。该协议在病理学研究中的效用通过显着增加的 CD45 阳性 (CD45+ ) 糖尿病小鼠模型肾脏中的细胞。CD45 和损伤标记物 KIM-1 的双重染色显示损伤的肾小管细胞周围积累了 CD45 +细胞。相邻组织切片上的 CD45 和 F4/80 巨噬细胞染色显示 CD45 +细胞与其他炎症细胞重叠。

[背景] CD45 是一种泛白细胞蛋白,具有酪氨酸磷酸酶活性,参与调节造血过程中的信号转导。它属于酪氨酸磷酸酶家族的受体型蛋白。它被严重糖基化并在有核造血细胞上高水平表达,包括粒细胞、淋巴细胞、巨噬细胞/组织细胞、肥大细胞、单核细胞、嗜碱性粒细胞和浆细胞。

临床上,CD45主要用于确认炎症细胞的存在和肿瘤的造血性质。在基础研究中,CD45 被广泛用作流式细胞术和组织学研究中的泛白细胞标记物。

由于小鼠组织中 CD45 的 IHC 检测方法引起了科学界的广泛兴趣,因此我们在本手稿中详细分享了我们的优化方案(Zheng等,2016)。我们展示了来自具有已知 CD45 阳性 (CD45 + ) 细胞的组织的图像,并专注于糖尿病小鼠的肾脏,我们证明糖尿病诱导的 CD45 表达显着增加,该表达与肾脏中的其他生物标志物共定位。

关键字:CD45, 糖尿病, 肾脏, KIM-1, 免疫组化, OVE

材料和试剂

 

1.     VWR微型盖板玻璃VWR,目录号:48393-081

2.     Microslides Superfrost Plus,目录号:48311-703VWRUSA

3.     ImmEdge 矢量,目录号:H-4000

4.     一抗:

a.     CD45(血管蛋白质组学,目录号:mAP-0058;物种:大鼠;目标:小鼠;稀释:1:400

b.     Kim-1R&D Systems,目录号:AF1817;物种:山羊;目标:小鼠;稀释:1:500

c.     F4/80Serotec,目录号:MCA497R,物种:大鼠;目标:小鼠;稀释:1:100

5.     二抗:

a.     抗大鼠,小鼠吸附(载体,目录号:BA-9401;物种:山羊;目标:大鼠;稀释:1:200;共轭:生物素化)

b.     抗山羊(Jackson Labs,目录号:705-095-147;物种:驴;目标:山羊;稀释:1:100;共轭:FITC

c.     抗大鼠(Jackson Labs,目录号:712-095-150;物种:驴;目标:大鼠;稀释:1:100;共轭:Cy3

6.     目标检索溶液10 ×浓缩;Dako,目录号:S1699

7.     30% H Sigma-Aldrich,目录号:216763-500 ml

8.     亲和素/生物素封闭试剂盒Vector Laboratories目录号:SP-2001ZF0917

9.     Vectastin Elite ABC 试剂盒过氧化物酶(标准),目录号:PK-6100ZF1011

10.  DAB底物试剂盒50×DAB ChromogenABCAM,目录号:ab64238

11.  山羊血清Jackson ImmunoResearch,目录号:005-000-121

12.  驴血清Jackson Immunoresearch,目录号:017000121

13.  苏木精溶液(Merck,目录号:1.05175.0500

14.  福尔马林10% 缓冲福尔马林;Fisher Scientific,目录号:23-427098

15.  试剂二甲苯纯化,目录号:UX-78923-75Cole-Parmer 

16.  VectaMount 永久安装介质;矢量,目录号:H-5000

17.  VECTASHIELD HardSet 带有 DAPI 的防褪色封固剂;Vector Laboratories目录号:H-1500

18.  二水柠檬酸三钠(Sigma,目录号:S4641

19.  乙二胺四乙酸二钠盐二水合物EDTA-Na )(Sigma,目录号:E5134

20.  氢氧化钠颗粒(EMD,目录号:SX0593

21.  柠檬酸盐缓冲液(见配方

22.  14% EDTA(见配方)

23.  PBS 中的 0.1% Tween 20(见配方)

24.  2.5% 正常山羊血清(见食谱)

25.  2.5% 正常驴血清(见食谱)

26.  PBS-T(见食谱)

 

设备

 

1.     显微镜(NikonECLIPSEE600 ZEISSAXIOIMAGER.A1 荧光显微镜)

2.     培养箱:StainTray 20 Place with Black Lid, Newcomer Supply Inc. # 6847-20BL ( https://www.newcomersupply.com/product/staintray-20-place/ )

3.     染色罐(Coplin Staining Jar with CoverWheatonVWR,目录号:25460-000 

4.     热板(搅拌器和热板搅拌器,Benchmark Scientific,目录号:H-4000-HS

 

程序

 

A.    组织切片

使用的载玻片来自小鼠组织,特别是骨髓、肺、肝和肾。除小鼠胫骨骨髓在福尔马林固定和石蜡包埋之间脱钙外,所有组织均采用标准福尔马林固定、石蜡包埋和组织块切片。为此,胫骨从正常成年 C57/Bl6 小鼠中分离出来,用福尔马林固定 48 小时,在 14% EDTA 溶液中脱钙 72 小时,然后用石蜡包埋。从糖尿病 OVE26 小鼠 [一种具有早期和持续性高血糖的转基因小鼠品系 (Epstein et al ., 1989),并鉴定为糖尿病肾病的可靠小鼠模型 (Zheng et al ., 2004)] 和单肾切除的 OVE26 小鼠的肾脏(郑2011)。非糖尿病对照肾获自年龄匹配的相同品系 FVB 小鼠。这些动物已经过白蛋白尿测试,并且已经用标准苏木精和伊红 (H & E) 和马森三色染色对它们的肾脏进行了组织学评估。具有明显肾小管间质纤维化的肾脏用于证明CD45染色。肾脏、肝脏和肺的组织切片以 4 µm 切割,胫骨切片以 5 µm 切割。切片安装在带正电的载玻片上。染色前,将所有石蜡切片在 70°C 下加热 30 分钟。骨髓组织被认为是阳性对照,省略了 CD45 一抗的切片被用作阴性对照。

 

B.    CD45染色

免疫组织化学 (IHC)

1.     对组织进行脱蜡和再水化:在二甲苯中对组织切片进行脱蜡,然后用分级乙醇将其再水化为水。(详细说明:在二甲苯中更换 3 次,每次 5 分钟;在 100% ETOH 中更换两次,每次 3 分钟;然后是 95% ETOH90% ETOH 80% ETOH,每次 3 分钟。然后,将载玻片移到水中,每次 3 分钟,有两个变化。)

2.     2阻断内部过氧化物酶活性:在室温 (RT) 下,用 3% H /PBS浸泡 Coplin 罐中的载玻片,20 分钟。

3.     PBS 清洗载玻片:将载玻片在 PBS 中浸泡 5 分钟,三个变化。

4.     在选择的缓冲液中提取抗原。对于该协议,抗原修复在 pH 695-100°C20 分钟的柠檬酸盐缓冲液中进行。[详细说明:将 50 ml 1×柠檬酸缓冲液加入玻璃科普林罐中,并将此罐置于加热板上的水浴烧杯中。一旦水浴沸腾,插入所述组织滑入科普林罐(最大幻灯片编号:10)。20 分钟后,将烧杯从加热板上取下,停止加热。在室温下将 Coplin 罐冷却 30 分钟。]

5.     PBS 清洗幻灯片 5 分钟 3 次。

6.     在载玻片上的组织切片周围画一个疏水圆圈:使用 ImmEdge ®疏水屏障 PAP 笔在组织切片周围画圈,形成耐热、防水的屏障,使试剂保持在组织标本上,并防止切片之间的试剂混合。

7.     在室温下用正常血清封闭组织切片 30 60 分钟(在此步骤和步骤 10 中添加了亲和素/生物素封闭程序)。

a.     PBS 中制备 2.5% 的正常山羊血清。

b.     将来自亲和素/生物素封闭试剂盒的亲和素溶液添加到 2.5% 的正常山羊血清中(每 1 毫升 4 滴),混合均匀。

c.     将载玻片放入孵化箱中。

d.     50-100 µl 封闭血清(含抗生物素蛋白溶液)涂抹在每个组织切片上。

e.     RT 中孵育切片 30 60 分钟。

8.     PBS-T 清洗载玻片,每次 5 分钟 3 次。

9.     应用一抗**

a.     制备抗体稀释剂:将生物素封闭液加入 2.5% 山羊血清/PBS(每 1 毫升 4 滴)中。

b.     用准备好的稀释剂稀释 CD45 抗体 1:400

c.     将稀释的抗体涂抹在组织切片上。

d.     4°C 下将孵育箱中的载玻片孵育过夜。

  1. PBS-T 清洗幻灯片 5 分钟 3 次。
  2. 应用二抗:

a.     通过在 PBS 中以 1:200-1:400 稀释生物素化山羊抗大鼠(小鼠吸收)IgG 来制备抗体。

b.     适用于组织切片,并在室温下将湿盒中的载玻片孵育 60 分钟。

  1. PBS-T 5 分钟洗涤五次。
  2. 应用 ABC 解决方案***

a.     VECTASTAIN Elite ABC 试剂盒中制备 ABC 试剂:将两滴(100 μl)试剂 A 加入 5 ml PBS 中并混合;然后在上述试管中加入两滴(100 μl)试剂 B 并立即混合。

b.     在每个组织切片上加入 50-100 µl ABC 试剂,室温孵育 30 分钟。 

  1. PBS-T 5 分钟洗涤 3 次。
  2. 应用 DAB

a.     通过将 200 µl 50 × DAB Chromogen 10 ml DAB 底物缓冲液混合来制备 DAB 试剂。

b.     DAB 应用于组织切片最多 10 分钟。

  1. 在水中清洗幻灯片 5 分钟 3 次。
  2. 如果需要,用苏木精复染细胞核。
  3. 用水 5 分钟清洗载玻片 3 次。
  4. 用乙醇脱水,然后用二甲苯脱水。(详细说明:在 90% ETOH 中孵育一次,在 100% 乙醇中孵育两次,每次 1 分钟,然后在二甲苯中孵育三次,每次 3 分钟。)
  5. 将永久性安装介质涂在载玻片上并盖上盖玻片。
  6. 在显微镜下观察载玻片,检查 DAB 的棕色是否在阳性对照中形成,以及阴性对照部分(省略一抗)是否没有颜色。
  7. 如果染色良好,请检查目标切片:

a.     存在阳性染色。

b.     阳性染色的位置(实质或间质组织)。

c.     记录在选定放大倍数下成像的组织切片的病理变化。如有必要,进行定量分析。让观察者对组标识视而不见。

笔记:

a.     **对于 CD45 F4/80 的共定位染色,选择具有三个相同对齐的连续切片的组织载玻片。CD45 F4/80 抗体分别应用于相邻部分。第三部分没有任何一抗。所有其他步骤都相同。

b.     *** 使用前让 ABC 试剂静置 15-30 分钟。

 

免疫荧光 (IF)CD45 KIM-1 双染色

1.     组织脱蜡和再水化:组织载玻片在二甲苯中脱蜡,然后用分级乙醇再水化成水。

2.     在选择的缓冲液中提取抗原。对于该协议,抗原修复在 pH 695-100°C20 分钟的柠檬酸盐缓冲液中进行。(详细说明:将 50 ml 1×柠檬酸缓冲液加入玻璃 Coplin 罐中,并将此罐置于加热板上的水浴烧杯中。水浴沸腾后,将组织玻片插入 Coplin罐(最大载玻片数量:10)。20 分钟后,从加热板上取下烧杯,停止加热。在室温下冷却 Coplin 30 分钟。)

3.     PBS 清洗幻灯片 5 分钟。

4.     在载玻片上的组织切片周围画一个疏水圆圈:用 ImmEdge ®疏水屏障 PAP 笔在组织切片周围画圈,以形成耐热、防水的屏障,以保持试剂位于组织标本上,并防止切片之间的试剂混合。

5.     血清阻断:

a.     PBS 中制备 2.5% 的正常驴血清。

b.     应用 50-100 µl 封闭血清覆盖组织切片。

c.     RT 中孵育幻灯片 60 分钟。

6.     PBS-T 短暂冲洗载玻片 3 分钟 3 次。

7.     应用一抗:

a.     制备混合抗体溶液:将 5 µl CD45 抗体(1:400)和 4 µl KIM-1 抗体(1:500)加入 2 ml 2.5% 驴血清/PBS

b.     将此混合抗体溶液应用于指定的组织切片。

c.     仅将免疫前血清应用于将用作阴性对照的部分。

d.     4°C 下过夜孵育载玻片。

8.     PBS-T 5 分钟清洗幻灯片 3 次。

9.     应用二抗:

a.     通过将 Cy3 偶联的抗大鼠 IgG FITC 偶联的抗山羊 IgG(均以 1:100 稀释)添加到 PBS 中并混合均匀,制备抗体混合物。

b.     将此混合抗体应用于组织切片。

c.     RT 的黑暗中孵育幻灯片 60 分钟。

  1. PBS-T 在黑暗中清洗幻灯片 5 分钟五次。
  2. 使用带有 DAPI 的安装介质安装组织切片,覆盖玻璃盖,并将载玻片存放在 4°C 的黑暗中。
  3. 使用荧光显微镜检查载玻片以验证阳性和阴性染色。在本研究中,DAPI/FITC/TRITC 过滤器用于显示细胞核 (DAPI)KIM-1 (FITC) CD45 (Cy3) 染色。
  4. 验证 IF 方法后,检查目标部分的阳性染色和位置。在选定的放大倍数下,通过具有多通道采集设置的 DAPIFITC TRITC 过滤器依次捕获样品图像,并使用软件合并免疫荧光图像。保持背景和曝光相等以进行图像比较。如有必要,进行定量分析。让观察者对组标识视而不见。

数据分析

 

A.    验证小鼠组织中的 CD45 抗体。

成功的 IHC 染色需要足够的抗体敏感性和特异性。CD45 是一种跨膜细胞表面糖蛋白,由绝大多数血淋巴谱系细胞表达。因此,骨髓似乎是一个很好的阳性对照组织。骨髓切片需要脱钙过程。在本研究中,小鼠胫骨骨髓用 14% EDTA 脱钙 72 小时。结果(图 1)表明 CD45 抗原性在脱钙后得以保留。如图 1A 所示,骨髓中的许多有核细胞对 CD45 染色呈阳性。当有核细胞位于红细胞 (RBC) 岛内时,CD45 +细胞(实线箭头)尤为明显(图 1C)。阳性染色与阴性染色的细胞形成对比,例如巨核细胞(血小板的前体)、脂肪细胞和周围的骨组织。当省略 CD45 抗体时,染色呈阴性(图 1B)。这些结果表明,优化的方案使用这种 CD45 抗体产生了灵敏和特异的染色。

 

 

1. 来自骨髓、肺和肝脏的正常成年小鼠组织切片的代表性 CD45 染色。

正常骨髓含有许多 CD45 +细胞 (A),如果省略 CD45 抗体,这些细胞就不存在 (B)。在更高的放大倍数 (C) 下,CD45 阳性骨髓细胞群(实线箭头)似乎不包括红细胞、巨核细胞(空心箭头)、脂肪细胞或周围的骨组织。在正常肺 (D) 和肝 (E) 中,CD45 在少量广泛分布的细胞中表达,可能是肺泡间隔中的巨噬细胞和肝窦中的库普弗细胞。染色是在福尔马林固定的石蜡包埋组织切片中进行的,骨骼的厚度为 5 µm,肺和肝脏的厚度为 4 µm。图像是在 20 倍或 40 倍放大率下拍摄的。

 

当来自正常小鼠肝脏或肺的组织切片被染色时,我们观察到阳性细胞单独分散在组织中。除了血管中的白细胞外,独特的 CD45 +细胞还存在于肺的肺泡隔或肝脏的血窦中。这些阳性细胞很可能是肺中的肺泡巨噬细胞或肝脏中的库普弗细胞。小鼠抗人 CD45 抗体已经很好地说明了人肺和肝脏中类似的 CD45 染色。https://www.novusbio.com/PDFs3/NBP2-52868.pdf

总体而言,该抗体方案经验证可检测正常小鼠骨髓、肺和肝脏中的CD45 +细胞。基于这些结果,我们继续在正常和糖尿病小鼠肾脏中进行 CD45 染色。

 

B.    正常和糖尿病小鼠肾脏中的 CD45 染色

将相同的 CD45 染色方案应用于正常和 OVE 糖尿病小鼠的肾脏。如图 2 所示,在低背景下显示细胞中明显的阳性染色。如同在骨髓中一样,如果省略 CD45 抗体,则在肾脏切片中没有看到染色(图像未显示)。在正常肾脏中,CD45 +细胞均匀分布在整个肾皮质 (A),主要分布在肾小管间质空间 (C)CD45 +细胞在正常肾脏中不形成簇。OVE 糖尿病会产生严重的蛋白尿(Zheng等人2004 年和 2016 年),这与 CD45 染色的流行和模式的急剧变化相吻合。糖尿病肾中 CD45 阳性细胞的数量明显增加(B)。它们的分布也发生了显着变化:CD45 +细胞倾向于围绕扩张或塌陷的小管(B D),在那里它们形成大而紧密的簇,似乎扩大了间质空间(E F)。这些结果清楚地表明,这种 CD45 染色方案在小鼠肾脏中运作良好,可用于分析糖尿病引起的主要变化。

 

2.正常和糖尿病肾中 CD45 +细胞的代表性图像。

在正常肾脏中,CD45 +细胞分布于整个肾皮质(A)。正常肾脏的更高放大倍数显示 CD45 +细胞主要位于 1 2 个阳性细胞组中的肾小管间质空间 (C)。在糖尿病肾中,CD45 +阳性细胞的数量大大增加,并且分布不均匀(B)。糖尿病肾中的大多数 CD45 +细胞位于扩张或塌陷的小管周围的小管间质空间中(B D)。CD45 +细胞紧密堆积成大簇,似乎扩大了间隙空间(E F)。石蜡包埋的福尔马林固定组织切片来自具有明显蛋白尿的正常 FVB OVE 糖尿病小鼠。CD45 染色按照方法部分中的描述进行,并通过 DAB 颜色产生进行可视化。A B 中的图像使用 20 倍物镜拍摄,CDE F 中的图像使用 100 倍物镜拍摄。 

 

 

3. OVE 糖尿病肾的典型图像,用 CD45 KIM-1 (ABC) 双染色或在连续切片上染色,用于 CD45 和巨噬细胞标记 F4/80 (DEF) 的共定位。

CD45 KIM-1 使用方法中描述的免疫荧光方案进行双染色。一抗用 CD45 Cy3(红色)和 KIM-1 FITC(绿色)可视化。肾损伤分子 KIM-1 主要出现在扩张的肾小管上皮细胞的顶端表面(A,原始放大倍数 20 倍,B C,原始放大倍数 40 倍)。CD45 +浸润细胞存在于 KIM-1 染色小管附近的小管间质中。通过将抗体分别应用于 IHC 中一张载玻片上的三个连续组织切片,将 CD45 的染色模式与单肾切除糖尿病小鼠肾脏切片中的 F4/80 进行比较;CD45 (D)F4/80 (E) 和无一抗对照 (F)CD45 F4/80 染色肾皮质的相同区域。DE F 的原始放大倍数为 20 倍。

 

C.    CD45 染色与 KIM-1 F4/80 染色在糖尿病肾中的比较

为了扩大我们对 CD45 染色增加的病理意义的理解,我们将其与 KIM-1 F4/80 染色进行了比较(图 3)。肾损伤分子-1 (KIM-1) 是一种在急性或慢性纤维化肾病中表达的上皮磷脂酰丝氨酸受体(Humphreys2013Mori2021)。KIM-1 CD45 通过方法中描述的免疫荧光方案在同一切片上进行双染色。一抗用 FITC(绿色)显示 KIM-1Cy3(红色)显示 CD45KIM-1 在扩张的肾小管上皮细胞的顶端表面表达 ( 3A-C)CD45 +浸润细胞簇存在于靠近许多被 KIM-1 染色的小管的小管间质中。KIM-1 CD45 染色之间的关联表明白细胞被受损的近端上皮吸引,进一步说明了糖尿病肾小管细胞损伤与肾脏炎症之间的相关性。 

F4/80 是一种广泛使用的巨噬细胞标记物。由于CD45F4/80抗体均来自大鼠,在同一切片中无法区分。因此,通过分别应用抗体来封闭连续组织切片来比较它们的染色模式。如图 3D E 所示,它们的染色模式非常相似,这意味着许多 CD45 +细胞是巨噬细胞。该策略可与其他感兴趣的抗体一起使用。

在本协议中,我们专注于描述 CD45 染色的程序,直到定量分析的可选程序。虽然我们之前在此 CD45 染色方案(Zheng2016)之后成功地进行了定量分析,但不同组中的染色经常如此明显,以至于无需定量即可得出结论。

 

概括

总之,我们优化了小鼠肾脏 CD45 染色的 IHC 方案。

由于一抗来自大鼠,因此选择生物素化的山羊抗大鼠(小鼠吸附)二抗进行染色。为了减少非特异性结合,使用了小鼠吸附抗大鼠 IgG (H+L) 产品,因为小鼠组织可能含有内源性小鼠免疫球蛋白。生物素化抗体可以确保最大程度的标记,而不会影响抗体的特异性或亲和力。

一些组织在切片中有内源性生物素或生物素结合蛋白、凝集素或非特异性结合物质,可能与亲和素、生物素化辣根过氧化物酶或其他亲和素/生物素系统成分结合,导致使用ABC试剂时产生高背景。因此,我们应用了亲和素-生物素封闭来减少背景染色。我们在本协议中采用了另一种方法来进行抗生物素蛋白/生物素阻断。将抗生物素蛋白和生物素封闭纳入血清封闭和一抗孵育步骤。该程序在 ABC 检测系统中保持低背景方面运行良好。

需要抗原揭露程序来实现 CD45 的最佳 IHC 染色,因为这种抗原可能会被长时间的福尔马林固定和石蜡包埋过程掩盖。在该协议中,使用了柠檬酸缓冲液 (pH 6.0) 热诱导表位检索 (HIER) 。在柠檬酸盐缓冲液中在 95-100 °C 处理 20 分钟的载玻片产生了足够的结果。来自商业来源或实验室制造的抗原修复解决方案都很好(配方如下)。

CD45 是一种泛白细胞蛋白,可用于识别造血谱系的细胞,如粒细胞、淋巴细胞和巨噬细胞/单核细胞。许多这些细胞通常分布在血流和一些组织中,如肺或肝脏,但特别富含淋巴结或脾脏。这些是方便的阳性对照组织。当小鼠胫骨用 14% EDTA 脱钙 72 小时时,骨髓组织也可以给出可靠的阳性染色。当省略一抗时不存在阳性染色。通过该协议,我们证明了糖尿病小鼠肾脏中 CD45 +细胞的浸润显着增加。用其他抗体对相邻切片进行双重染色或染色可以扩大我们进一步将炎症细胞浸润与肾脏疾病进展中的其他有害因素联系起来的能力。

食谱

 

1.     柠檬酸盐缓冲液(10 mM 柠檬酸,pH 6.0

2.94 g 二水柠檬酸钠溶解在 800 ml Milli-Q 水中,调节至 pH 6.0,最后用 Milli-Q 水定容至 1,000 ml

2.     14% 乙二胺四乙酸

要制备 2,000 毫升 14% EDTA,将 250 EDTA-2Na 加入 1,750 毫升 Milli-Q 水中,然后加入 25 克氢氧化钠颗粒。继续搅拌并稍微加热,直到 EDTA 溶解。将 pH 值调至 7.0,然后用水将体积调至 2,000 毫升。

3.     2.5% 正常山羊血清

250 µl 正常山羊血清添加到 10 ml PBS 中。

4.     0.1% 吐温 20 PBS

0.1 ml Tween 20 添加到 100 ml PBS

5.     2.5% 正常驴血清

250 µl 正常驴血清添加到 10 ml PBS 中。

6.     PBS-T

0.1% 吐温 20 PBS

 

致谢

 

该协议是从以前的出版物(Zheng2016改编和扩展的。我们感谢 JDRF NIH 的赠款支持。JDRF 3-2005-932 SZJDRF 1-2005-88 NIDDK DK072032 PE

 

利益争夺


作者没有要披露的利益冲突或竞争利益。

 

伦理

 

所有动物程序均遵循 NIH 实验动物护理和使用指南,并获得路易斯维尔大学机构动物护理和使用委员会的批准。

 

参考

 

1.     Epstein, PN, Overbeek, PA Means, AR (1989)钙调蛋白诱导的转基因小鼠早发性糖尿病。 单元格58(6)1067-1073

2.     Humphreys, BD, Xu, F., Sabbisetti, V., Grgic, I., Movahedi Naini, S., Wang, N., Chen, G., Xiao, S., Patel, D., Henderson, JM, et艾尔(2013)慢性上皮肾损伤分子 1 表达导致鼠肾纤维化。 J Clin Invest 123(9): 4023-4035

3.     Mori, Y., Ajay, AK, Chang, JH, Mou, S., Zhao, H., Kishi, S., Li, J., Brooks, CR, Xiao, S., Woo, H.(2021)KIM-1 介导肾小管细胞摄取脂肪酸以促进进行性糖尿病肾病。细胞代谢表 33(5)1042-1061 e1047

4.     Zheng, S.Coventry, S.Cai, L.PowellDWJalaVRHaribabu, B. Epstein, PN (2016)通过遗传缺失糖尿病 OVE 小鼠的非典型趋化因子受体 ACKR2 来保护肾脏。J 糖尿病研究20165362506

5.     Zheng, S., Noonan, WT, Metreveli, NS, Coventry, S., Kralik, PM, Carlson, EC Epstein, PN (2004)OVE26 糖尿病小鼠晚期糖尿病肾病的发展。糖尿病53(12)3248-3257

6.     Zheng, S.Huang, Y.Yang, L.Chen, T.Xu, J. Epstein, PN (2011)糖尿病 OVE26 小鼠的单肾切除术大大加速了蛋白尿、纤维化、炎症细胞浸润和基因表达的变化。 Nephron Exp Nephrol 119(1): e21-32

登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zheng, S. and Epstein, P. N. (2021). CD45 Immunohistochemistry in Mouse Kidney. Bio-protocol 11(22): e4230. DOI: 10.21769/BioProtoc.4230.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。