The ovarian paraffin sections were deparaffinized, rehydrated, and subjected to high temperature (95–98 °C) antigen retrieval with 0.01% sodium citrate buffer (pH 6.0) for 16 min. After that, the sections were washed with PBS on a shaker for 5 min, and then blocked with 1% normal donkey serum in PBS for 1 h at room temperature and incubated with primary antibodies for 12–16 h at 4 °C. The antibodies used were listed in Table S1 . Subsequently, ovarian sections were rinsed thoroughly with PBS and incubated with Alexa Fluor 488- or 555-conjugated donkey secondary antibodies for 1 h at 37 °C. Then slides were rinsed in PBS, counter-stained with Hoechst 33342 for 5 min. Finally, sealed the sections in an anti-fade fluorescence mounting medium with coverslips. Immunohistochemistry was performed using Histostain™-SP Kits (PV-9001, ZSGB-BIO, China) and DAB peroxidase substrate kits (ZLI-9017, ZSGB-BIO, China) according to the manufacturer's protocols. Sections were examined and photographed using Nikon 80i or Nikon A1 laser.
Dab peroxidase substrate kit
Manufactured by ZSGB-BIO
Sourced in China
The DAB peroxidase substrate kit is a laboratory reagent used for the detection of peroxidase enzyme activity in various applications, such as immunohistochemistry and immunocytochemistry. The kit provides a 3,3'-diaminobenzidine (DAB) solution that serves as a chromogenic substrate for peroxidase, producing a brown color reaction that can be visualized under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Histostain sp kit, by ZSGB-BIO (2 mentions)
Pv 9001, by ZSGB-BIO (2 mentions)
A1 laser, by Nikon (1 mentions)
C1210, by Applygen (1 mentions)
Ab217344, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Gb11093, by Wuhan Servicebio Technology (1 mentions)
Arg22476, by Arigo Biolaboratories (1 mentions)
Ab238132, by Abcam (1 mentions)
Citrate edta antigen retrieval solution, by Beyotime (1 mentions)
A1002, by ABclonal (1 mentions)
Ap0489, by ABclonal (1 mentions)
Optical microscope, by Olympus (1 mentions)
Ab79823, by Abcam (1 mentions)
Ba0752, by Boster Bio (1 mentions)
Ba0290, by Boster Bio (1 mentions)
Ba0407, by Boster Bio (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Apc cd4 rm4 5, by BioLegend (1 mentions)
Collagenase hyalurinidase, by STEMCELL (1 mentions)
11 protocols using dab peroxidase substrate kit
1
Ovarian Immunohistochemistry Protocol
2
Immunofluorescence and Immunohistochemistry of Ovaries
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Freshly isolated ovaries were fixed in cold 4% paraformaldehyde overnight, dehydrated in gradient alcohol, embedded in paraffin, and sectioned at 5 μm. Ovarian sections were deparaffinized, rehydrated and subjected to antigen retrieval with 0.01% sodium citrate buffer (pH 6.0) at high temperature (95–98 °C) for 16 min. The sections were then rinsed thoroughly with PBS and blocked with normal donkey serum in PBS for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies used are listed in Table S1 . Next, ovarian sections was rinsed thoroughly with PBS and incubated with Alexa Fluor 488- or 555- conjugated secondary antibody for 1 h at 37 °C. The ovarian sections were rinsed thoroughly with PBS, stained with DAPI for 5 min and sealed in anti-fade fluorescence mounting medium (C1210, Applygen, China) with coverslips. Immunohistochemistry was performed using Histostain™-SP Kits (PV-9001, ZSGB-BIO, China) and DAB peroxidase substrate kits (ZLI-9017, ZSGB-BIO, China) according to the manufacturer’s protocols. Sections were examined and photographed using Nikon 80i or Nikon A1 laser.
3
Ovarian Follicle Quantification and Immunohistochemistry
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Ovaries in the diestrous stage were removed and fixed in 10% formalin solution at room temperature overnight with rotation, dehydrated in an ascending series of ethanol solutions (70%, 80%, 90%, 95%, and 100%), and then embedded in paraffin. The ovaries were serially sectioned at a thickness of 5 μm and subjected to HE staining for follicle counting. The number of follicles in distinct stages containing oocytes with a clearly visible nucleus per ovary was counted according to our previous report.9 (link) For immunochemistry, 3 μm-thick sections were deparaffinized and dehydrated with xylene and an ascending series of alcohol solutions. After antigen retrieval, the sections were incubated with primary antibodies (Table S2) at 4°C overnight, incubated with biotin-labelled secondary antibodies for 30 min at room temperature, and developed using a DAB peroxidase substrate kit (Zsbio, Beijing, China).
4
Immunohistochemical Analysis of Ephrin-A1 and Microvessel Density
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The immunohistochemical analysis was performed as previously described, with some modifications30 (link). Paraffin-embedded tissues were incubated with anti-ephrin-A1 antibody (catalog no. ab199697, Abcam, Cambridge, MA). Subsequently, the sections were stained using a polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and were visualized with a DAB peroxidase substrate kit (ZLI-9017, ZSGB-BIO, Beijing, China). The immunostained images were captured using a microscope (LEICA DMi8). Protein expression levels were analyzed by calculating the integrated optical density (IOD) per stained area using Image-Pro Plus version 6.0.
Anti-CD34 antibody (BA3414, Boster, Wuhan, China) was used to identify endothelium31 (link),33 (link), and the MVD quantification was performed in accordance with the counting method of Weidner et al.34 (link). Briefly, the five most vascularized areas with the greatest number of tumor microvessels were chosen at low power fields (100× magnification). The microvessels were then counted on a 200× high power field within the designated neovascular region. The MVD was defined as the number of vessels per high power field (200× magnification).
Anti-CD34 antibody (BA3414, Boster, Wuhan, China) was used to identify endothelium31 (link),33 (link), and the MVD quantification was performed in accordance with the counting method of Weidner et al.34 (link). Briefly, the five most vascularized areas with the greatest number of tumor microvessels were chosen at low power fields (100× magnification). The microvessels were then counted on a 200× high power field within the designated neovascular region. The MVD was defined as the number of vessels per high power field (200× magnification).
5
Immunohistochemistry of Immune Cell Markers
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Paraffin sections were dewaxed in xylene and rehydrated in ethanol, respectively. Antigen retrieval was performed in a microwave oven for 2 min at high power and then for 10 min at low power, followed by cooling down to room temperature. The activity of endogenous peroxidase was quenched with 3% H2O2 for 10 min. Tissue blocking was performed with goat serum for 1 h at room temperature. Tissue sections were then incubated at 4°C over-night with primary antibodies for T lymphocytes (CD3, ARG52744, Arigo), helper T lymphocytes (CD4, ab183685, Abcam), cytotoxic T lymphocytes (CD8, ab217344, Abcam), Treg lymphocytes (FOXP3, GB11093, Servicebio), natural killer cell (CD49b, AGR57601), or macrophages (F4/80, ARG22476, Arigo). The sample were then incubated with horseradish peroxidase labeled secondary antibody (1: 500) for 1 h at 37°C. Color development was performed using the DAB peroxidase substrate kit (ZSGB-BIO) according to the manufacturer’s instruction. The sections were finally dehydrated in ethanol and xylene, and sealed with neutral resin. The image signal recorded by microscope was converted into digital signal by Image J to analyze the distribution and staining intensity of positive markers.
6
Immunohistochemical Profiling of Cardiac Markers
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For immunohistochemical staining (IHC), heart tissue sections were subjected to antigen retrieval with citrate-EDTA antigen retrieval solution (P0086; Beyotime, China) in a 95–100 °C water bath for 15 min after dewaxing and rehydration. The subsequent steps included inhibiting endogenous peroxidase activity and permeabilization with 0.1% Triton X-100 solution for 15 min. After being blocked with 5% bovine serum albumin at room temperature for 30 min, the sections were incubated overnight at 4 °C with the following primary antibodies: Ly6G (1:500, ab238132, Abcam), HMGB1 (1:350, ab79823, Abcam), YAP (1:200, A1002, ABclonal), and phospho-YAP-S127 (1:100, AP0489, ABclonal). The sections were thoroughly washed with PBS, and subsequently incubated with an HRP-labeled goat anti-rabbit IgG secondary antibody (PV-6001; ZSGB-bio, Beijing, China) for 20 min at 37 °C. A DAB Peroxidase Substrate Kit (ZLI-9018; ZSGB-bio, Beijing, China) was used to visualize the positive region. Following hematoxylin staining of the nucleus, the sections were subjected to ethanol dehydration and xylene clearing. The sections were imaged with an optical microscope (Olympus, Tokyo, Japan) and then analyzed using ImageJ (NIH V1.8.0.112).
7
IBDV Colonization in ILP Cells
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In order to detect whether the IBDV colonized in the ILP cells at the early stage, the duodenum and cecal tonsil from the infected birds were collected. Sections of the cecal tonsils and duodenum were then fixed in 10% formalin solution and prepared as previously described [9 (link)]. The IBDV antigen was detected using a monoclonal anti-IBDV-VP2 antibody (a kind gift from Professor Aijian Qin, Yangzhou University, Yangzhou, Jiangsu, China) at a dilution of 1:10,000. The secondary anti-mouse IgG HRP-labeled antibodies were used according to the manufacturer’s instructions. DAB (DAB peroxidase substrate Kit, ZSGB-bio, Beijing, China) was used to visualize the enzyme-linked complex. Sections were observed by light microscopy.
8
Immunohistochemical Quantification of Synovial Angiogenesis
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The immunohistochemical analysis was performed as previously described with some modifications[34 (link)]. Knee joint sections on slides were incubated with anti-TSP-1, TGF-β1, CTGF, VEGF and von Willebrand factor (vWF) antibody (BA2130, BA0290, BA0752, BA0407, BA0046, Boster, Wuhan, China). Subsequently, the sections were stained using polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and were visualized with DAB peroxidase substrate kit (ZLI-9017, ZSGB-BIO, Beijing, China). Rabbit anti-mouse vWF was used to identify endothelium as described before [41 (link)]. Following immunostaining, the synovium area in the joint of each section was evaluated under a microscope (LEICA DMi8) in three randomly selected areas at a magnification of 400×. The number of positively stained cells and total cells were counted by two different observers, and the means of the ratio of these two groups of cells were calculated. The number of microvessels positively stained with anti-vWF was also counted in the same area on the next serial section.
9
Histological Evaluation of Arthritis in Mice
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After being sacrificed, the knee joints of mice were collected and stained with HE staining. To quantitatively evaluate the severity of arthritis, a scoring system was employed referring to the reported protocol (Zhang et al., 2017 (link)). Histological changes were examined by microscopic evaluation and scored in a blinded manner by two independent observers.
The immunohistochemical analysis was performed as previously described with some modifications (Zhang et al., 2017 (link)). Knee joint sections on slides were incubated with anti-FOXO1 and anti-VEGF antibodies (Wanleibio, Shenyang, China). Subsequently, the sections were stained using the polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and were visualized with DAB Peroxidase Substrate Kit (ZLI-9017, ZSGB-BIO, Beijing, China). Following immunostaining, the synovium area in the joint of each section was evaluated under a microscope (LEICA DMi8) in three randomly selected areas at a magnification of 100×. Image-Pro Plus 6 (Media Cybernetics, Inc.) was used to analyze the average integrated optical density (IOD) according to a previously described protocol (Wang et al., 2019 (link)).
The immunohistochemical analysis was performed as previously described with some modifications (Zhang et al., 2017 (link)). Knee joint sections on slides were incubated with anti-FOXO1 and anti-VEGF antibodies (Wanleibio, Shenyang, China). Subsequently, the sections were stained using the polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and were visualized with DAB Peroxidase Substrate Kit (ZLI-9017, ZSGB-BIO, Beijing, China). Following immunostaining, the synovium area in the joint of each section was evaluated under a microscope (LEICA DMi8) in three randomly selected areas at a magnification of 100×. Image-Pro Plus 6 (Media Cybernetics, Inc.) was used to analyze the average integrated optical density (IOD) according to a previously described protocol (Wang et al., 2019 (link)).
10
Immunohistochemical and Flow Cytometric Analysis of Tumor-Infiltrating Lymphocytes
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Fresh tumor tissues were fixed in 4 % paraformaldehyde solution for 48 hours, and the tissues were dehydrated and embedded in paraffin. Immunohistochemistry (IHC) analysis of the embedded tissue was performed in 5 mm thick sections. In brief, primary antibodies, 4-HNE (1:400, ab46545, Abcam) were incubated overnight at 4 C. Staining was performed using Rabbit polymer assay system (ZSGB-BIO) and DAB peroxidase substrate Kit (ZSGB-BIO). Images (5 per tumor) were randomly acquired from the slice using an Axio Zoom.V16 microscope (Agilent) at 400 3 magnification.
Tumor infiltration lymphocyte analysis 4T1 xenografts isolated from BALB/c mice were sliced and digested with collagenase/hyalurinidase (Stemcell Technologies, Vancouver, BC, Canada) and DNase (Sigma). Lymphocytes were enriched using HistopaqueÒ-1083 (Sigma) and then T cells were enriched using the Dynabeads untouched mouse T cell kit (Invitrogen). The isolated T cells were fixed with 4 % paraformaldehyde for 5min and stained with PE-CD3ε (145-2C11; Biolegend), PE-Cyanine7-IFN-g (XMG1.2; Biolegend), APC-CD4 (RM4-5; Biolegend) and FITC-CD8a (53-6.7; BD PharmingenTM) at room temperature for 15 min in the dark. After three times washed, the populations of infiltration T cells were detected and analyzed with BD FACS (LSRF Fottessa) cytometer.
Tumor infiltration lymphocyte analysis 4T1 xenografts isolated from BALB/c mice were sliced and digested with collagenase/hyalurinidase (Stemcell Technologies, Vancouver, BC, Canada) and DNase (Sigma). Lymphocytes were enriched using HistopaqueÒ-1083 (Sigma) and then T cells were enriched using the Dynabeads untouched mouse T cell kit (Invitrogen). The isolated T cells were fixed with 4 % paraformaldehyde for 5min and stained with PE-CD3ε (145-2C11; Biolegend), PE-Cyanine7-IFN-g (XMG1.2; Biolegend), APC-CD4 (RM4-5; Biolegend) and FITC-CD8a (53-6.7; BD PharmingenTM) at room temperature for 15 min in the dark. After three times washed, the populations of infiltration T cells were detected and analyzed with BD FACS (LSRF Fottessa) cytometer.
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