Immunohistochemical staining was performed using 3′,3′-diaminobenzidine (DAB) according to the peroxidase complex method. Paraffin sections were dehydrated at 44 °C for 60 min and deparaffinized. The antigen was activated with citrate buffer at pH 9.0 (#415201; Nichirei, Tokyo, Japan) and blocked using 10% horse serum (#16050; Gibco, Gaithersburg, MD, USA) and 1% sodium azide. Sections were incubated with polyclonal rabbit anti-human ITGA7 (1:400, #HPA008427; Abcam) at 4 °C overnight. The samples were then washed and stained using Histofine simple stain MAX PO (Nichirei) as the secondary antibody. Color development was acquired with DAB (#347-00904; Dojindo).
3 3 diaminobenzidine (dab)
Manufactured by Dojindo Laboratories
Sourced in Japan, United States
3,3′-diaminobenzidine is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) for the detection and visualization of target antigens. It produces a brown reaction product upon enzymatic conversion.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated anti rabbit igg, by Vector Laboratories (3 mentions)
Abc method, by Vector Laboratories (3 mentions)
Histofine simple stain max po, by Nichirei Biosciences (2 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (2 mentions)
Ax80 microscope, by Olympus (2 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Abc elite, by Vector Laboratories (2 mentions)
Mayer s hematoxylin, by Fujifilm (2 mentions)
Eis elements software, by Nikon (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Bz 9000, by Keyence (2 mentions)
Envision system, by Agilent Technologies (2 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Biotinylated horse anti mouse igg, by Vector Laboratories (1 mentions)
Envision system hrp labeled polymer anti rabbit, by Agilent Technologies (1 mentions)
Hpa006539, by Merck Group (1 mentions)
Envision system hrp, by Agilent Technologies (1 mentions)
Avidin biotin peroxidase complex kit, by Vector Laboratories (1 mentions)
Ab71916, by Abcam (1 mentions)
Ca 94010, by Vector Laboratories (1 mentions)
26 protocols using 3 3 diaminobenzidine (dab)
1
Immunohistochemical Detection of ITGA7
2
Immunohistochemical Detection of Synaptopodin in Rat Kidney
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The cryostat sections of rat kidney were rinsed in PBS and then boiled in 10 mM citrate buffer, pH 6.0, for 10 min to retrieve the antigenic sites. After cooling to RT, the sections were incubated in methanol containing 0.3% H2O2 for 30 min to block endogenous peroxidase activity. After several rinses in PBS, the sections were incubated in 5% normal horse serum (NHS)/1% BSA in PBS for 10 min to block nonspecific binding and then incubated with a mouse monoclonal antibody against synaptopodin (clone G1D4; Progen Biotechnik, Heidelberg, Germany; diluted 1:50 with 5% NHS/1% BSA in PBS) at 4 °C overnight. As controls, the procedure was performed without primary antibodies. After the sections were washed with PBS, they were incubated with biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA; diluted 1:200 with 1% BSA in PBS) at RT for 40 min, followed by washing with PBS. Then, sections were incubated in a freshly prepared solution of avidin-biotinylated HRP complex (ABC) kit (Vector Laboratories; diluted 1:50 with 1% BSA in PBS) for 30 min. After PBS washes, the peroxidase reaction was developed by incubating in 0.05% DAB (Dojindo Laboratories, Kumamoto, Japan) in 0.05 M Tris buffer, pH 7.6, containing 0.001% H2O2 for 8 min.
3
ANGPTL2 and GLUT3 Immunohistochemistry
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Formalin‐fixed, paraffin‐embedded specimens were cut into 4‐μm sections and deparaffinized. Sections were autoclaved with citrate buffer (pH 6.0) for antigen retrieval. Sections were incubated with 3% H2O2 for 5 minutes to block endogenous peroxidase activity and then incubated with anti‐ANGPTL2 Ab and anti‐GLUT3 (1:100, HPA006539; Sigma‐Aldrich), diluted with Block Ace (KAC) at 4°C overnight. After washing with PBS, sections were incubated for 30 minutes with EnVision+ System‐HRP‐labeled Polymer Anti‐rabbit (Dako) for visualization with DAB (Dojindo). Slides were counterstained 20 seconds with hematoxylin.
4
Immunohistochemical Analysis of Tissue Microarrays
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IHC analyses of tissue microarrays were performed, as described previously [16 (link)]. Briefly, samples were deparaffinized in xylene and dehydrated in a graded series of ethanol. After rinsing in TBS buffer, the samples were processed for antigen retrieval in a 1 mM EDTA retrieval solution (pH 9.0) (Nichirei Co.) at 95 °C for 40 min. Endogenous peroxidase was then blocked by incubation in 0.3% H2O2-methanol at room temperature for 10 min. After rinsing with TBS, samples were incubated with primary antibodies against human PD-L1 (1:200) and p-FAK (1:100) for 30 min, and then with EnVision (Dako Corp) for 30 min. After rinsing in TBS, the coloring reaction was performed with DAB (Dojindo) for 5 min. Each sample was counterstained with hematoxylin. These processes were all performed at room temperature. For sirius red staining, tissue slides were dewaxed, dehydrated, and stained with Picro-sirius red for 1 h (Sigma-Aldrich, S365548). After staining, slides were washed in two dips of acidified water and dehydrated with 100% ethanol. Finally, slides were cleared in xylene and mounted. Quantification of collagen staining was performed using Image J software [7 (link)].
5
Immunohistochemical Analysis of Brd4, c-Myc, and p53
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Immunostaining was performed to observe the expression patterns of Brd4, c-Myc, and p53 in the cytological and histological specimens. All slides were subjected to antigen retrieval in a microwave oven, with a maximum strength of 1000 W, using EDTA (pH 8.0), for 20 min followed by incubation with either a rabbit polyclonal anti-p53 antibody (1:50 dilution at room temperature (RT, 24±2˚C) for 2 h; clone ab131442; Abcam, Cambridge, MA, USA), a rabbit monoclonal anti-c-Myc antibody (1:100 dilution at RT for 2 h; clone ab32072; Abcam), or a rabbit monoclonal anti-BRD4 antibody (1:100 dilution at RT for 2 h; clone ab128874; Abcam). The slides were then washed and incubated with the Envision+/HRP system (Dako, Glostrup, Denmark). Immunoreactive cells were visualized using DAB (Dojindo, Kumamoto, Japan) and counterstained with hematoxylin.
6
Immunohistochemical Detection of Cubn
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Embryo samples were fixed in 4.0% PFA/PBS. Fixed samples were washed in PBS, then reacted with a primary antibody (anti-Cubn) at 4°C overnight, and then reacted with biotinylated anti-rabbit IgG (Vector Laboratories) at room temperature for 2 h. Samples were analyzed with the avidin–biotin–peroxidase complex kit (Vector Laboratories), and visualized with 0.05% DAB (Dojindo Laboratories, Kumamoto, Japan) and 0.01% hydrogen peroxide in 50 mmol l−1 Tris buffer (pH 7.2) at room temperature for 10 min. The procedure for electron microscopy is described in our previous study (Iida et al., 2019 (link)).
7
Immunostaining of Intestinal Tissue
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For immunostaining, intestinal tissues were fixed in 4% paraformaldehyde in PBS at 4°C for overnight and then were embedded in optimal cutting temperature compound (OCT) (Sakura Finetek). 7-µm-thick frozen sections were boiled in 50 mM sodium citrate buffer solution (pH=6.0) for antigen retrieval, then were blocked using 1% BSA in PBS at room temperature for 1 hour. Subsequently, the sections were incubated with primary antibodies in PBS with 1% BSA at 4°C for overnight, and then incubated with secondary antibodies in PBS with 1% BSA at room temperature for 1 hour. The primary antibodies included rabbit anti-EpCAM (1:200; ab71916; Abcam), rabbit anti-mouse IgA secondary antibody (1:1000; NB7506; Novus) and rabbit anti-Claudin-7 (1:200; 34-9100; Thermo Fisher Scientific, Inc.). Immunohistochemical analysis was performed with biotin-conjugated goat-anti-rabbit secondary antibody (JAC-111-065-144, Jackson ImmunoResearch), HRP-ABC complex (CA 94010, VECTASTAIN) and DAB (LF778, DOJINDO Laboratories), and immunofluorescence analysis was performed with Alex Fluor 488-labeled secondary antibodies (Invitrogen). The immunostaining images were observed using the PerkinElmer Automated Quantitative Pathology System.
8
Immunohistochemistry and Histological Analysis of NAFLD
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For immunohistochemistry, endogenous peroxidase activity was blocked using 0.03% H2O2 in Methanol. The sections were incubated with the optimal dilutions of anti-F4/80 (AbD Serotec, Oxford, UK), ER-TR7 (Abcam, Cambridge, MA, USA) antibodies overnight at 4 °C. After incubation with appropriate secondary antibodies, substrate reaction was performed using 3,3′-Diaminobenzidine (Dojindo, Kumamoto, Japan) solution.
NAFLD activity score was calculated according to Kleiner et al.20 (link). For qualitative assessment of Oil Red O, positive areas were scored for 5 grades by microscopy. For quantitative analysis of F4/80, ER-TR7 and Sirius red positive areas, bright field images of stained sections were captured using a digital camera (DP72, Olympus, Tokyo, Japan) around central veins at 400-fold magnification, and the positive areas in 5 fields were measured using WinROOF image processing software (Mitani, Tokyo, Japan). The results were determined as the means of five different fields of each section.
NAFLD activity score was calculated according to Kleiner et al.20 (link). For qualitative assessment of Oil Red O, positive areas were scored for 5 grades by microscopy. For quantitative analysis of F4/80, ER-TR7 and Sirius red positive areas, bright field images of stained sections were captured using a digital camera (DP72, Olympus, Tokyo, Japan) around central veins at 400-fold magnification, and the positive areas in 5 fields were measured using WinROOF image processing software (Mitani, Tokyo, Japan). The results were determined as the means of five different fields of each section.
9
Quantification of Cell Proliferation Markers
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Tissue samples were fixed with 4% formalin and embedded in paraffin. The slices were cut to 5 μm thickness. IHC was performed by blocking the rehydrated tissue sections using bovine serum overnight at 4°C, and subsequently applying the Anti-Ki67 antibody (Abcam, Cat. no. ab15580) and Anti-PCNA antibody (Abcam, Cat. no. ab92729). The sections were washed and then subjected to incubation with biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (Vector Laboratories, CA, USA). The ABC method (Vector Laboratories, CA, USA) was employed along with 3,3’- diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) as a substrate to detect the staining. The sections were visualized using an AX-80 microscope (Olympus, Tokyo, Japan), and Image J software (http://imagej.nih.gov/ij/ ) was employed for image analysis and quantification of positive expression. Statistical analysis was conducted using One-Way ANOVA.
10
Immunohistochemical Analysis of Renal Fibrosis
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