Axonal microtubules and Schwann cells were visualized using immunohistochemical staining with anti-neurofilament and anti-S-100 antibodies, respectively. Briefly, the rehydrated sections were blocked using normal goat serum (Vector ABC Elite Kit; Vector Laboratories) for 1 h, incubated with rabbit anti-neurofilament and anti-S-100 primary antibodies (1:500; Abcam, Cambridge, UK) overnight at 4 °C, reacted with biotinylated goat anti-rabbit IgG (Vector ABC Elite Kit) for 2 h at RT, reacted with the avidin–biotin peroxidase complex (Vector ABC Elite Kit) for 1 h at RT, and finally developed using diaminobenzidine substrate (DAB kit; Vector Laboratories). The relative staining intensities, average positive cell sizes, and average axonal diameters were analyzed using the ImageJ software. The parameters are expressed as the mean ± standard error (n = 3/group).
Diaminobenzidine dab substrate kit
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
Diaminobenzidine (DAB) substrate kit is a laboratory reagent used for chromogenic detection in immunohistochemistry and immunocytochemistry. It provides a brown-colored precipitate at the site of the antigen-antibody reaction, allowing for visualization of the target protein.
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Lab products found in correlation
Vectastain elite abc kit, by Vector Laboratories (4 mentions)
Rabbit anti neurofilament antibody, by Abcam (3 mentions)
Vector elite abc kit, by Vector Laboratories (2 mentions)
Blocking reagent, by Vector Laboratories (2 mentions)
Vectastain elite abc rabbit kit, by Abcam (2 mentions)
Mouse anti human p16, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti human p21, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse or anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Streptavidin biotin hrp complex, by GE Healthcare (1 mentions)
Tween 20, by Merck Group (1 mentions)
Spot rt3 camera, by Nikon (1 mentions)
Hoechst 33342 dye, by Merck Group (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Harris modified hematoxylin solution, by Merck Group (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
14 protocols using diaminobenzidine dab substrate kit
1
Visualizing Axonal Microtubules and Schwann Cells
2
Visualizing Axonal Microtubules and Schwann Cells
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Axonal microtubules and Schwann cells were visualized via immunohistochemistry using anti-neurofilament and anti-S-100 antibodies, respectively. Briefly, the rehydrated sections were blocked with normal goat serum (Vector ABC Elite Kit; Vector Laboratories) for 1 h, incubated with rabbit anti-neurofilament and anti-S-100 primary antibodies (1:500; Abcam, Cambridge, UK) overnight at 4℃, reacted with biotinylated goat anti-rabbit IgG (Vector ABC Elite Kit) for 2 h at room temperature (RT), reacted with avidin-biotin peroxidase complex (Vector ABC Elite Kit) for 1 h at RT, and developed with diaminobenzidine substrate (DAB kit; Vector Laboratories). The relative staining intensities, average positive cell sizes, and average axonal diameters were analyzed using the ImageJ software (https://imagej.nih.gov/ij/download.html ). The parameters are expressed as mean±error (SEs) (n= 5/group).
3
Detection of DNA Damage in Tissue Sections
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TUNEL staining was designed to detect DNA double-strand damage according to the manufacturer’s protocol (Roche). A diaminobenzidine (DAB) substrate kit (Vector laboratories) was used for peroxidase staining, and the sections were then counter stained with hematoxylin. PANT staining was performed for the detection of cells with single-strand DNA damage. Slices were rinsed in 1% Triton-X-100 to permeabilize the sections and then 2% H2O2 to quench endogenous peroxidases. Afterwards, the sections were incubated with the PANT reaction mixture (10 mM 2-mercaptoethanol, 20 μg/ml BSA, 20 μM each of dGTP, dCTP and dTTP; 1 μM dATP, 19 μM biotinylated dATP and 40 units/ml of DNA polymerase I) at 37 °C for 90 min. Biotinylated dATP incorporated into damaged DNA was detected by incubation with streptavidin-horseradish peroxidase and DAB substrate.
The number of Iba-1-positive cells, Fluoro-Jade C-positive cells, TUNEL-positive cells and PANT-positive cells per field were counted by an investigator who was blinded to the experimental conditions. All of these cells were calculated randomly in five separate fields in four different slices/per animal.
The number of Iba-1-positive cells, Fluoro-Jade C-positive cells, TUNEL-positive cells and PANT-positive cells per field were counted by an investigator who was blinded to the experimental conditions. All of these cells were calculated randomly in five separate fields in four different slices/per animal.
4
Immunohistochemical Analysis of Cell Cycle Regulators
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5-μm sections of paraffin embedded, formalin fixed, lung were stained for p16, p21, and p53 by immunohistochemistry as previously described. [23 (link)] Briefly, the sections were incubated in PBS containing 5% normal goat serum and 1% bovine serum albumin (BSA), then incubated with a 1:100 dilution of mouse anti-human p16 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human p21 (Santa Cruz Biotechnology) or mouse anti-human p53 antibody (Santa Cruz Biotechnology) overnight at 4°C. The sections were washed, incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology). After washing, the bound peroxidase activity was detected using a diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA).
5
Quantification of Ovarian AMH Expression
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Ovaries from young (n = 4) and aged adult (n = 4) C57Bl6/J mice (separate to the main study cohort) were immersion-fixed in Bouin's fixative overnight, wax embedded, and sectioned at 5 μm thickness. The xylene-dewaxed sections were rehydrated through graded ethanol solutions and incubated in blocking solution (0.1 μg/mL in phosphate-buffered saline with 5%, normal donkey serum (Merck, Cat# D9663), 0.08% w/v Tween20 (Sigma-Aldrich, Cat# P1379) and 1% w/v bovine serum albumin) for 20 minutes. Goat anti-human AMH C-20 antibody (Santa Cruz, Cat# sc-6886, Santa Cruz, CA, USA, RRID: AB_649207) was applied at 0.1 μg/mL in phosphate-buffered saline with 0.08% w/v Tween20 (Sigma-Aldrich, Cat# P1379, Kenilworth, NY, USA) and 1% w/v bovine serum albumin, overnight incubation at 4 °C. Biotin-SP-AffiniPure Donkey Anti-Goat IgG (H + L), (Jackson ImmunoResearch Lab, Cat# 705-065-003, West Grove, PA, USA, RRID: AB_2340396) was applied at 2 µg/mL for 1 hour at room temperature followed by streptavidin-biotin-HRP complex (GE Healthcare, Cat#RPN1051, Chicago, IL, USA) diluted 1:100 for 1 hour at room temperature. Chromogen staining was generated with diaminobenzidine (DAB substrate kit, Vector Labs, Cat# SK-4100, Newark, CA, USA) with hematoxylin counterstain.
6
Immunohistochemical Analysis of Tissue Samples
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Dorsal skin or tumor samples were fixed in 4% paraformaldehyde in PBS, dehydrated in ethanol, embedded in paraffin, and sectioned at 5 μm. For immunofluorescence detection, sections were deparaffinized, rehydrated, and subjected to antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0). After blocking, sections were stained with primary antibody at 4 °C overnight in a humid chamber, followed by incubation with the appropriate fluorochrome-conjugated secondary antibody for 2 h at room temperature. Nuclei were stained with Hoechst 33342 dye (Sigma, St. Louis, MO, USA). Immunohistochemistry was performed on paraffin-embedded sections subjected to antigen retrieval as described above. After blocking, sections were incubated with primary antibodies at 4 °C overnight in a humid chamber, followed by incubation with biotinylated IgG at RT for 30 min. Detection of the signal was carried out using Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA, USA) according the manufacturer’s instructions. The sections were counterstained with hematoxylin. Microscopy was performed using a fluorescence microscope (Nikon Eclipse E600), Spot RT3 camera and Spot imaging software.
7
Immunohistochemical Analysis of Xenograft Tumors
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Immunohistochemistry was performed on the tumor samples from the pCMV/HCT 116 and AKT/HCT 116 xenografts. Tumors were fixed and stained with hematoxylin and eosin for pathological examination. Tumor tissue slides were dewaxed and rehydrated before antigen retrieval. They were then incubated with primary antibodies against Ki67, phosphorylated AKT, p65, and Notch1 followed by incubation with respective horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Diaminobenzidine (DAB Substrate Kit, Vector Laboratories, Vernon Hills, IL, USA) was used for coloration, and a dark brown color was considered to be positive staining. The Ki76 score was essentially defined as the percentage of total number of tumor cells with nuclear staining and tumors are classified as low, intermediate, and highly proliferating35 .
8
Xenograft Tumor Growth Assay in Nude Mice
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Six-week-old female BALB/c nu/nu athymic mice (Shanghai Slac Experimental Animals Co., Shanghai, P.R. China) were used for experimental tumorigenesis assay. During the study, the mice were maintained in pathogen-free conditions under controlled temperature and humidity with 12-h light/dark cycle, and supplied with standard rodent food and water ad libitum. For the xenograft tumor growth assay, equivalent amount of miR-206-overexpressing C666-1 cells (approximately 1 × 107) and their control cells were inoculated subcutaneously into the axilla of the nude mice. Tumor volume during the experiment was measured weekly with slide calipers, and volumes were estimated according to the standard formula V = (length × width2)/2. After 3 weeks, the mice were euthanized, and the tumors were exercised and weighed. The 4% paraformaldehyde-fixed tumor tissues were embedded in paraffin, then sectioned at 5-μm thickness, and used for immunohistochemistry for Ki-67 or caspase 3, and reaction was developed with a diaminobenzidine (DAB) substrate kit (Vector Laboratories, Inc., Burlingame, CA, USA). All experimental protocols for animal study were approved by the Ethics Committee for Animal Research at the Fudan University.
9
Immunohistochemical Analysis of Vascular and Inflammatory Markers
10
Immunohistochemical Analysis of MKRN2 in Tumor Tissues
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5 μm thick paraffin-embedded tumor tissues were pretreated with 4% formaldehyde. Paraffin-embedded tissue sections were prepared followed by dewaxed, rehydrated, antigen repaired, and endogenous peroxidase activity blocked as well serum blocking. The sections were incubated at 4° C overnight with the primary antibody anti-MKRN2 (1:100), then incubated at 37° C for 1h with the secondary antibody horseradish peroxidase (HRP)-conjugated antibodies (DACO, Kyoto, Japan). The results were investigated by Diaminobenzidine (DAB) Substrate Kit (Vector Laboratories, Inc., Burlingame, CA).
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