Kidney sections were boiled in citric acid buffer (10 mM, pH 6.0) for 20 min for antigen retrieval. The sections were blocked in 10% normal horse serum and incubated with a primary antibody (1:100 diluted) against Wilms’ Tumor Protein 1 (WT1) from Boster Biological Technology (Pleasanton, CA, USA) overnight at 4 °C. The sections were incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min and then developed using a 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories) for 30 s. The sections counterstained with hematoxylin were analyzed by using a CKX41 light microscope (Olympus). The number of stained nuclei was counted from 10 images of 200× microscopic fields per kidney section from each group.
Avidin biotin peroxidase complex solution abc solution
Manufactured by Vector Laboratories
Sourced in United States
Avidin-biotin-peroxidase complex solution (ABC solution) is a laboratory reagent used in immunohistochemistry and other biotechnology applications. It consists of avidin, biotin, and horseradish peroxidase, which form a stable complex. The ABC solution is designed to amplify signal detection in these techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ckx41 light microscope, by Olympus (8 mentions)
Biotinylated secondary antibody, by Vector Laboratories (7 mentions)
3 3 diaminobenzidine dab peroxidase substrate kit, by Vector Laboratories (5 mentions)
Wilms tumor protein 1 wt1, by Boster Bio (2 mentions)
Egr1 antibody, by Cell Signaling Technology (1 mentions)
Ab61392, by Abcam (1 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine peroxidase substrate kit, by Vector Laboratories (1 mentions)
Ab48506, by Abcam (1 mentions)
Hematoxylin and eosin h e, by Merck Group (1 mentions)
Bp 9200, by Vector Laboratories (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Mca771ga, by Bio-Rad (1 mentions)
8 protocols using avidin biotin peroxidase complex solution abc solution
1
Immunohistochemical Analysis of WT1 in Kidney
2
Immunohistochemical Analysis of Kidney Tissue
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The 10% formalin-fixed and paraffin-embedded kidney tissue sections (5 μm-thick) were prepared. Briefly, the fixed kidney sections were deparaffinized, rehydrated and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. The sections were blocked in 10% normal horse serum and incubated with a primary antibody against CD68 (Cluster of Differentiation 68) from (Abcam) or Wilms’ Tumor Protein 1 (WT1) from Boster Biological Technology (Pleasanton, CA, USA) overnight at 4 °C. The sections were incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution, Vector Laboratories) for 30 min and developed using 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories). Then, the sections were counterstained with Mayer’s hematoxylin and analyzed using a CKX41 light microscope (Olympus). The number of CD68-stained cells or WT1-stained nuclei (equivalent to the number of podocytes) was counted from 10 images of 400× magnification per kidney section from each group, (n = 3), using ImageJ software (NIH).
3
Immunohistochemical Analysis of Ly-6B.2 and Egr-1
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The fixed tissue sections were deparaffinized, rehydrated, and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide, and nonspecific binding was blocked with 10% normal horse serum. The sections were incubated with a primary antibody against Ly-6B.2 antibody (Bio-Rad, Hercules, CA, USA) or Egr-1 antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, and then with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in an avidin–biotin–peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min, and developed using a 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories). Then, the sections were counterstained with hematoxylin, and the images were obtained using a CKX41 light microscope (Olympus).
4
Nitrotyrosine Immunohistochemistry in Liver Sections
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Immunohistochemical staining was performed as previously described [3 (link)]. In brief, paraffin liver sections (5 µm) were deparaffinized, rehydrated, and antigen retrieved in sodium citrate buffer (10 mM, pH 6.0; iNtRON Biotechnology, Seongnam, Korea) for 20 min. The sections were blocked in 10% normal horse serum (Vector Laboratories, Burlingame, CA, USA) and incubated with a primary antibody for nitrotyrosine (ab61392, Abcam, Cambridge, UK) overnight at 4 °C. Sections were washed and incubated with a biotinylated secondary antibody (Vector Laboratories) for 1 h at room temperature. Sections were washed again and incubated in avidin–biotin–peroxidase complex solution (ABC solution; Vector Laboratories) and then developed using a 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories). The sections were counterstained with hematoxylin and analyzed using a CKX41 light microscope (Olympus, Tokyo, Japan).
5
Immunohistochemical Analysis of GOLPH3
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The 10% formaldehyde-fixed, paraffin-embedded liver or kidney tissue sections were prepared. Briefly, the fixed tissue sections were deparaffinized, rehydrated, and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. Subsequently, the sections were permeabilized with 0.3% Triton X-100 for 10 min in PBS. The sections were blocked using 10% normal horse serum and incubated with a primary antibody against GOLPH3 (Proteintech Group, Chicago, IL, USA) at 4 °C overnight. Next day, the sections were washed and incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were then incubated in the avidin-biotin-peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min, and labeled by adding 3,3’-diaminobenzidine (DAB) Peroxidase Substrates (Vector Laboratories), being counterstained with Mayer’s Hematoxylin. Finally, the sections were mounted and examined using a CKX41 light microscope (Olympus). GOLPH3-positive cells were analyzed and counted using ImageJ software (NIH, Bethesda, MD, USA).
6
Quantifying Podocytes and Autophagy in Kidney
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The 10% formaldehyde-fixed and paraffin-embedded kidney tissue sections (5 μm-thick) were prepared. Briefly, the fixed kidney sections were deparaffinised, rehydrated and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. The sections were blocked in 10% normal horse serum and incubated with a primary antibody against Wilms' tumour protein 1 (WT1) from Boster Biological Technology (Pleasanton, CA, USA) or LC3B from Sigma (St. Louis, MO, USA) overnight at 4 °C. The sections were incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min and developed using 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories). Then, the sections were counterstained with Mayer's haematoxylin and analysed using a CKX41 light microscope (Olympus). The number of stained nuclei was counted (equivalent to the number of podocytes) from 10 images of 200× of HPF per kidney section from each group (n = 3). LC3B-positive cells were counted in the kidney sections stained by LC3B, from 10 images of 400× magnification per section from each group (n = 3) using ImageJ software (NIH).
7
Histological Analysis of Fixed Liver Samples
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The fixed livers were processed for paraffin embedding and sectioned to 5-μm thickness. Sections were stained with Hematoxylin and Eosin (H&E) (Sigma-Aldrich, St. Louis, MO, USA) by a standard protocol.
For 4-Hydroxynonenal (4-HNE) immunehistochemical staining, sections were boiled in citric acid buffer (10 mM, pH 6.0) for 20 min for antigen retrieval. After blocking in 10% normal horse serum, sections were incubated with 4-HNE antibody (ab48506, Abcam) overnight at 4 °C. Sections were then incubated with a biotinylated secondary antibody (BP-9200, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Sections were washed and incubated in avidin–biotin–peroxidase complex solution (ABC solution; Vector Laboratories) and developed using a 3,3ʹ-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories). TUNEL assay was performed using an in-situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol. All images were captured using a CKX41 light microscope (Olympus, Tokyo, Japan).
For 4-Hydroxynonenal (4-HNE) immunehistochemical staining, sections were boiled in citric acid buffer (10 mM, pH 6.0) for 20 min for antigen retrieval. After blocking in 10% normal horse serum, sections were incubated with 4-HNE antibody (ab48506, Abcam) overnight at 4 °C. Sections were then incubated with a biotinylated secondary antibody (BP-9200, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Sections were washed and incubated in avidin–biotin–peroxidase complex solution (ABC solution; Vector Laboratories) and developed using a 3,3ʹ-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories). TUNEL assay was performed using an in-situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol. All images were captured using a CKX41 light microscope (Olympus, Tokyo, Japan).
8
Immunohistochemical Staining of Kidney and Brain
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The paraffin-fixed kidney sections were deparaffinized, rehydrated, and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. The kidney sections were blocked in 10% normal horse serum and incubated with a primary antibody for Ly-6B.2 (MCA771GA; Bio-Rad, Hercules, CA, USA) overnight at 4 °C. The free-floating brain sections were incubated with primary antibody for GFAP (Z033429; Agilent Dako, Santa Clara, CA, USA) overnight at 4 °C. After washing of unbound antibodies, the sections were incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Then, the sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min and developed by using a 3,3’-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA). Finally, the sections were counterstained with hematoxylin and analyzed using a CKX41 light microscope (Olympus, Tokyo, Japan).
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