The tissue slices were deparaffinized and rehydrated (detailed procedure as previously described in H&E staining). In order to retrieve antigen, samples were first immersed in 0.1 M citrate buffer at 96 °C for 10 min and later incubated in 5% BSA for 2 h. The slices were incubated with antibody against CD31 (1:100, Cat# ab281583, Abcam) and α-SMA (1:200, Cat# ab32575, Abcam) at 4 °C overnight. After rinsing with PBS and incubated with biotinylated goat anti-rabbit secondary antibody (Cat# PV-6001, ZSGB-BIO, China) for 2 h, the tissue slices were colored with 3,3-diaminobenzidine (DAB) (Cat# AR1022, Boster, China), stained with hematoxylin, dehydrated with a gradient ethanol series, soaked with xylene, and then sealed with resin. Finally, five random locations of each tissue slice were selected to count the new capillaries with endothelial cells positive for CD31 via microscope (400 ×) or to analyze the average optical density values for α-SMA expression at the indicated time points using Image-Pro Plus 6.0 Software.
3 3 diaminobenzidine dab
Manufactured by Boster Bio
Sourced in China
3,3′-diaminobenzidine (DAB) is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) to visualize the presence and location of target proteins or enzymes. It produces a brown insoluble precipitate upon oxidation, which can be observed under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Ab32575, by Abcam (1 mentions)
Ab281583, by Abcam (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by ZSGB-BIO (1 mentions)
Biotinylated secondary antibody, by Abcam (1 mentions)
Streptavidin biotin complex reagent, by Boster Bio (1 mentions)
Rabbit anti human collagen type 2 antibodies, by Merck Group (1 mentions)
Biotinylated goat anti rabbit igg, by Boster Bio (1 mentions)
Axioskop 40, by Zeiss (1 mentions)
Sodium citrate buffer, by Abcam (1 mentions)
Ocn antibody, by Abcam (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Strept avidin biotin complex, by Boster Bio (1 mentions)
Collagen 2, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Leagene (1 mentions)
Ab222782, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Proteintech (1 mentions)
Goat polyclonal anti iba1, by Abcam (1 mentions)
Mouse monoclonal anti neun, by Abcam (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Ds u3 camera interface, by Nikon (1 mentions)
15 protocols using 3 3 diaminobenzidine dab
1
Quantifying Angiogenesis and Smooth Muscle Actin
2
Immunohistochemical Analysis of PIK3C3 and CD133 Expression
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All slides were subjected to heat-induced epitope retrieval antigen procedure by heating in a microwave using citrate-based antigen retrieval solution. The slides were then exposed to primary antibodies at 4 °C overnight followed by incubation with biotinylated secondary antibody (DAB; Boster) at room temperature for 1 h. Reaction results were visualized by incubation with 3,3′-diaminobenzidine (DAB; Boster). The chromogenic reaction was stopped by washing with tap water, and the slides were then dehydrated in an ascending alcohol gradient, followed by cleared twice with xylene, and then mounted in neutral balsam. The percentage of positive staining areas were scored 0–4 (0, 1 [1–25%], 2 [26–50%], 3 [51–75%], and 4 [76–100%]) and staining intensity was scored 0–3 (0, negative; 1, weak; 2, moderate; and 3, strong). The overall protein expression score was calculated by multiplying the positivity and intensity scores ranging from 0 to 12. The antibodies, rabbit anti-human PIK3C3 (Abcam) and rabbit anti-human CD133 (Abcam) were used.
3
Immunohistochemical Evaluation of FAT4 in Gastric Tissue
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Formalin-fixed, paraffin-embedded gastric tissue samples were cut into 4-μm thick sections for immunohistochemical staining. Sections were incubated with rabbit polyclonal anti-FAT4 antibody (1 : 150, Novus Biologicals, Littleton, CO, USA) at 4 °C overnight, washed with PBS, and incubated with a biotinylated secondary antibody (Abcam, Cambridge, UK) for 30 min. Peroxidase reactivity was visualised with 3,3′-diaminobenzidine (DAB) (Boster, Wuhan, China) and counterstaining with haematoxylin. Evaluation of immunohistochemistry (IHC) was performed using a previously described method (Kusinska et al, 2009 (link)). The IHC intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The percentage of positive cells was scored as 0 (negative), 1 (<10%), 2 (10–50%), or 3 (>50%). The final score was obtained by multiplying the scores for IHC intensity by the percentage of positive cells. A final score of ⩽1 was considered as negative, and a final score of 2–9 was considered as positive.
4
Immunohistochemical Analysis of Cartilage Tissue
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Sections of cartilage nodule tissue were deparaffinized and rehydrated and then treated with 3% H2O2. Slides were next incubated in 0.01 M citrate buffer for 20 min at 94–98°C and blocked with 5% BSA. Primary antibodies included rabbit anti-human collagen type II antibodies (1:80, Sigma-Aldrich). The staining was visualized using a microscope (Axioskop 40, Zeiss) by applying streptavidin-biotin complex reagent (Boster) and 3,3′-diaminobenzidine (DAB; Boster) after the treatment with a solution of biotinylated goat anti-rabbit IgG (Boster). Sections were treated using the same process but without incubation with a primary antibody as the negative control.
5
Evaluating Bone Implant Integration
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The prepared slides were rehydrated, and immumohistochemistry staining (IHC) was applied to examine the protein expressed level of Collagen I (COL-I), Osteocalcin (OCN) and Bone morphogenetic protein-2 (BMP-2). Briefly, the rehydrated slides were heated in sodium citrate buffer (Abcam) for antigen retrieval. Then the slides were washed with PBS, followed by being exposed to 5% BSA for 1 h at 37 °C to block non-specific binding. After that the slides were incubated with primary antibodies at 4 °C overnight, followed by incubation with 50 μL of mouse-anti rabbit-horseradish peroxidase (HRP) (BOSTER) for 50 min at room temperature. The reaction products were visualized with 3-3′ diaminobenzidine (DAB) (BOSTER) according to the manufacturer's instructions. The antibodies were diluted as follows: OCN antibody (1: 500, Abcam), BPM-2 antibody (1: 200, Beyotime) and COL-І antibody (1: 200, Beyotime). Quantification data of IHC were performed by calculating the mean optical density value of positive-staining areas. The area of interest was firstly focused on the new formed issue between the host bone and cement implant, then areas (n = 5) with remarkably high immunohistochemical staining were selected and manually quantified using the Image-Pro Plus software (Media Cybernetics).
6
Immunohistochemical Analysis of Cartilage
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The sections for cartilage pellets were dewaxed and rehydrated and then incubated with 3% H2O2. After antigen retrieval, blocked with 5% bovine serum albumin (BSA, Leagene, China). The sections were incubated with primary antibody (Collagen II, 1 : 100, Cell Signaling Technology, USA; SOX9, 1 : 50, Santa Cruz Biotechnology, USA) overnight at 4°C. After washed with phosphate-buffered saline (PBS; Gibco, USA), the slides were incubated in secondary antibody (Boster, China), streptavidin-biotin complex, and 3,3′-diaminobenzidine (DAB; Boster, China) follow one another. The stained sections onto microscope slides were evaluated by light microscopy (Leica, Germany).
7
Immunostaining protocol for cell analysis
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For immunostaining, the sections were deparaffinized, briefly washed with 0.1 M PBS (pH 7.4), and incubated for 10 min in 3% H2O2 to quench endogenous peroxidase activity. Primary antibodies were applied overnight at 4 °C or incubated for 2 h at 37 °C. After being washed three times with PBS and incubated with goat-anti-rabbit HRP-conjugated secondary antibody for 1 h at 37 °C, an immunohistochemical staining signal was developed with 3, 3′-diamino-benzidine (DAB, BOSTER Biological Technology, Wuhan, China). The numbers of positive cells and relative intensity were counted using Image J software (National Institutes of Health, MD, USA).
8
Immunohistochemical Analysis of CD133 Expression
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Tumour tissues from the nude mice were fixed in 4% formaldehyde for 24 h, embedded in paraffin, and serially sectioned at a thickness of 6 μm. Sections were deparaffinized and stained with anti-CD133 (1:500; ab222782; Abcam) at 4 °C overnight, and incubated with the secondary antibody for 1 h at 37 °C. Reaction results were shown by incubation with 3, 3′-Diaminobenzidine (DAB; Boster). After washing with tap water to stop the chromogenic reaction, the sections were dehydrated in an ascending alcohol gradient, cleared twice with xylene and mounted in neutral balsam. Then, the sections were examined and imaged by microscope (Nikon).
9
Immunohistochemical Staining of c-kit and Tryptase
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Tissue was sectioned (6 μm) and stained using standard immunohistochemical techniques for microstructure analysis. Samples underwent deparaffinization in an ethanol gradient, followed by washing in PBS; endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS for 15 min at 37 °C. The sections were then covered with 5% bovine serum albumin (BSA). The sections were incubated with rabbit anti-c-kit (1:100) antibody (Boster Bio-Technology, Wuhan, China) and rabbit anti-tryptase (1:100) antibody (Boster Bio-Technology) overnight at 4 °C, as well as 1 section was incubated with PBS as a control. After incubation with primary antibody, the sections were incubated in biotinylated antirabbit immunoglobulin G secondary antibody (Boster Bio-Technology) for 1 h at 4 °C. The sections were then rehydrated in PBS and incubated with avidin–biotinylated peroxidase complex for 45 min at 37 °C. For color development, peroxidase activity was revealed using 3,3-diaminobenzidine ((DAB) Boster Bio-Technology) under a microscope.
10
Immunohistochemical Analysis of ESR2 and AR
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For IHC staining, renal sections were incubated at 4°C overnight with primary antibodies against ESR2 (1:50; Cloud-clone, Wuhan, Hubei, China) and AR (1:50; Cloud-clone, Wuhan, Hubei, China), and then washed with PBS. The specimens were subsequently incubated with an HRP-conjugated goat anti-rabbit antibody (1:500, Proteintech, Chicago, IL, United States) at 37°C for 1 h. Next, the sections were stained with 3,3′-diaminobenzidine (DAB; Boster, Wuhan, Hubei, China) for 6 min.
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