CD45 Immunohistochemistry in Mouse Kidney
小鼠肾脏中的 CD45 免疫组织化学
发布: 2021年11月20日第11卷第22期 DOI: 10.21769/BioProtoc.4230 浏览次数: 4144
评审: Xiaoyi ZhengAnonymous reviewer(s)
下载 PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Dec 2015
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
VWR micro cover glass (VWR, catalog number: 48393-081)
Microslides (Superfrost Plus, catalog number: 48311-703, VWR, USA)
ImmEdge pen (Vector, catalog number: H-4000)
Primary antibodies:
CD45 (Angio-Proteomie, catalog number: mAP-0058; Species: rat; Target: mouse; Dilution: 1:400)
Kim-1 (R&D Systems, catalog number: AF1817; Species: goat; Target: mouse; Dilution: 1:500)
F4/80 (Serotec, catalog number: MCA497R, Species: rat; Target: mouse; Dilution: 1:100)
Secondary antibodies:
Anti-rat, mouse adsorbed (Vector, catalog number: BA-9401; Species: goat; Target: rat; Dilution: 1:200; Comjugated: Biotinylated)
Anti-goat (Jackson Labs, catalog number: 705-095-147; Species: donkey; Target: goat; Dilution: 1:100; Comjugated: FITC)
Anti-rat (Jackson Labs, catalog number: 712-095-150; Species: donkey; Target: rat; Dilution: 1:100; Comjugated: Cy3)
Target Retrieval solution (10× concentrated; Dako, catalog number: S1699)
30% H2O2 (Sigma-Aldrich, catalog number: 216763-500 ml)
Avidin/Biotin Blocking Kit (Vector Laboratories, catalog number: SP-2001, ZF0917)
Vectastin Elite ABC Kit (Peroxidase (Standard), catalog number: PK-6100, ZF1011)
DAB substrate kit (50× DAB Chromogen; ABCAM, catalog number: ab64238)
Goat serum (Jackson ImmunoResearch, catalog number: 005-000-121)
Donkey serum (Jackson Immunoresearch, catalog number: 017000121)
Hematoxylin solution (Merck, catalog number: 1.05175.0500)
Formalin (10% Buffered Formalin; Fisher Scientific, catalog number: 23-427098)
Reagents Xylenes (Purified, catalog number: UX-78923-75, Cole-Parmer)
VectaMount (permanent Mounting medium; Vector, catalog number: H-5000)
VECTASHIELD HardSet (Antifade mounting medium with DAPI; Vector Laboratories, catalog number: H-1500)
Sodium citrate tribasic dihydrate (Sigma, catalog number: S4641)
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na2) (Sigma, catalog number: E5134)
Sodium hydroxide pellets (EMD, catalog number: SX0593)
Citrate buffer (see Recipes)
14% EDTA (see Recipes)
0.1% Tween 20 in PBS (see Recipes)
2.5% normal goat serum (see Recipes)
2.5% normal donkey serum (see Recipes)
PBS-T (see Recipes)
Equipment
Microscopes (Nikon, ECLIPSE, E600 and ZEISS, AXIO, IMAGER.A1 Fluorescent microscope)
Incubation box: StainTray 20 Place with Black Lid, Newcomer Supply Inc. # 6847-20BL (https://www.newcomersupply.com/product/staintray-20-place/)
Staining jar (Coplin Staining Jar with Cover, Wheaton; VWR, catalog number: 25460-000)
Hotplates (Stirrers and Hotplate Stirrers, Benchmark Scientific, catalog number: H-4000-HS)
Procedure
文章信息
版权信息
© 2021 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zheng, S. and Epstein, P. N. (2021). CD45 Immunohistochemistry in Mouse Kidney. Bio-protocol 11(22): e4230. DOI: 10.21769/BioProtoc.4230.
Download Citation in RIS Format
分类
免疫学 > 免疫细胞成像
免疫学 > 炎症性疾病
生物科学 > 生物技术
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
