For immunoperoxidase staining, after heat antigen retrieval, sections were treated with 3% hydrogen peroxide in water for 20 min, incubated with serum-blocking solution, and incubated overnight at 4°C with primary antibody (see Table S12 ). Following washing, sections were then sequentially incubated with biotinylated secondary antibody for 2 h at room temperature, avidin-biotin peroxidase (ABC, 1:100 Vector Laboratories) for 1 h at room temperature, and 3,3′-Diaminobenzidine (DAB) substrate solution (Vector Laboratories) for 5 min. Sections were washed, dried, and cover slipped with DPX mounting medium. Bright-field or fluorescence images were taken with an Olympus X71 microscope using appropriate filters.
3 3 diaminobenzidine dab substrate solution
Manufactured by Vector Laboratories
Sourced in United States
3,3'-Diaminobenzidine (DAB) substrate solution is a chromogenic substrate used in immunohistochemistry and immunocytochemistry to visualize the location of target antigens in biological samples. It produces a brown colored reaction product upon enzymatic conversion.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
X71 microscope, by Olympus (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Observer d1 microscope, by Zeiss (1 mentions)
Hematoxylin qs, by Vector Laboratories (1 mentions)
Axiovision imaging system software, by Zeiss (1 mentions)
Ly6g clone 1a8, by BioLegend (1 mentions)
5 bromo 2 deoxyuridine, by Roche (1 mentions)
Cell death detection kit, by Roche (1 mentions)
4 protocols using 3 3 diaminobenzidine dab substrate solution
1
Immunoperoxidase Staining Protocol
2
Immunohistochemical Analysis of Microglia
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Mice were anesthetized using isoflurane and then trans-cardially transfused with 0.1 M phosphate buffer (pH 7.4) and 4% paraformaldehyde (wt/vol), and the brains were removed. The brain tissues were fixed overnight at 4% paraformaldehyde solution, submerged in 30% sucrose solution at 4 °C, embedded with OCT medium (Tissue-Tek, cat#4583) and stored at − 80 °C. Brains were sectioned sagittally starting from the mid-cerebrum and immune-stained for Iba-1. After incubating with anti- Iba-1 (Wako, cat# 019-19741) at 4°C overnight, the sections were treated with biotin-SP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch labs, INC, Code# 111-065-003, PA, USA) for 1h, and with ABC solution prepared from VECTASTAIN Elite ABC Kit (Vector laboratories, Inc; cat# PK-6100, CA, USA) for 30 min followed by 3,3′-diaminobenzidine (DAB) substrate solution (Vector laboratories, Inc; cat# SK-4100) for visualizationn.
3
Immunohistochemical Analysis of Ly6G+ Cells
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Mice were euthanized 14 days after AC injection with Ad5E1 tumor, and their tumor-bearing eyes were processed for immunohistochemistry as previously described.30 The eye sections were incubated with a 1:500 dilution of primary rat antibody to Ly6G (clone 1A8; Biolegend, San Diego, CA, USA) and developed using biotinylated secondary antibody (Vectastain Elite ABC Kit; Vector Laboratories; Burlingame, CA, USA) and 3, 3′-diaminobenzidine (DAB) substrate solution (Vector Laboratories) and counterstained with hematoxylin QS (Vector Laboratories). Stained eyes were imaged using differential interference contrast (DIC) microscopy with a ×40 brightfield lens on the Zeiss Observer.D1 microscope with AxioVision Imaging System software (Carl Zeiss, Jena, Germany).
4
Assessing Proliferation and Apoptosis in Kidney Tissue
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Cell proliferation was assayed in paraffin-embedded kidney tissue by incorporation of 5-bromo-2-deoxyuridine (Roche Molecular Biochemicals, Mannheim, Germany), as previously described [29 (link)]. Pregnant mice received an intraperitoneal injection of BrdU (100 mg/g of body weight) 2 h prior to sacrifice. BrdU-positive cells were identified using an anti-BrdU peroxidase-conjugated antibody (Roche Molecular Biochemicals, Mannheim). Immunoreactivity was visualized using Aminoethyl Carbazole horseradish peroxidase chromogen/substrate solution (Vector, USA). Apoptosis was assessed in paraffin-embedded kidney tissue using the cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) and visualized using 3,3’-Diaminobenzidine (DAB) substrate solution (Vector, USA).
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