Tissues were first prepared for staining by fixing in 4% paraformaldehyde at room temperature for 10 min and then washed with PBS. After neutralization of the endogenous peroxidase with 3% H2O2 for 10 min, the sections were incubated with protein blocking buffer for 10 min before undergoing incubation with the primary antibody. Anti-SMO (E-5: sc-166685, Santa Cruz Biotechnology Inc., Dallas, TX) staining was developed using DAB (3,3′-Diaminobenzidine) (Vector Laboratories, Burlingame, CA) followed by hematoxylin counterstaining (MilliporeSigma, St. Louis, MO). H&E staining was also performed to access the location of the tumor (fig. S6). The SMO protein intensity inside the brain tumor was quantified using ImageJ.
Dab 3 3 diaminobenzidine
Manufactured by Vector Laboratories
Sourced in United States
DAB (3,3′-Diaminobenzidine) is a chromogenic substrate commonly used in immunohistochemistry and histochemical applications. It produces a brown-colored precipitate upon oxidation, allowing for the visualization of target antigens or enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain abc kit, by Vector Laboratories (3 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Hematoxylin counterstaining, by Merck Group (1 mentions)
Ab150078, by Abcam (1 mentions)
Ab101404, by Abcam (1 mentions)
Immpress micropolymer hrp conjugated anti rabbit igg, by Vector Laboratories (1 mentions)
Immpress micropolymer hrp conjugated anti mouse igg, by Vector Laboratories (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Vectastain elite abc avidin biotin complex kit, by Vector Laboratories (1 mentions)
Vectra polaris imaging system, by PerkinElmer (1 mentions)
Roti histokitt 2, by Carl Roth (1 mentions)
Axiocam hrc camera, by Zeiss (1 mentions)
Axioplan 2 imaging microscope, by Zeiss (1 mentions)
Bcl w, by Abcam (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
Cellsens, by Olympus (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Cx43 biological microscope, by Olympus (1 mentions)
Hm 325 rotary microtome, by Thermo Fisher Scientific (1 mentions)
Anti ho 1, by Abcam (1 mentions)
18 protocols using dab 3 3 diaminobenzidine
1
Immunohistochemical Analysis of SMO in Brain Tumors
2
Immunohistochemistry for Cancer Markers
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Anti-CXCL14 (rabbit IgG; NBP1-31398; Novus, Littleton, CO, USA), anti-CXCL16 (rabbit IgG; ab101404, Abcam, Cambridge, MA, USA), anti-CEA (mouse IgG1, clone II-7, M7072, Dako, Glostrup, Denmark) and FITC-conjugated anti-EpCAM (mouse IgG1, clone BerEP4, F0860; Dako) were used as primary antibodies. Mouse IgG, ready to use (Dako), rabbit IgG ready to use (Dako) and FITC-conjugated mouse IgG (F0313; Dako) were used as negative controls. ImmPRESS micropolymer HRP conjugated anti-rabbit IgG (MP-7401, Vector laboratories, Burlingame, CA, USA), ImmPRESS micropolymer HRP conjugated anti-mouse IgG (MP-7402, Vector laboratories), and Alexa Fluor 555-conjugated goat anti-rabbit IgG (ab150078, Abcam) were used as secondary antibodies. The peroxidase substrate used was DAB (3,3′-diaminobenzidine, Vector Laboratories).
3
Immunohistochemical Staining of FFPE Tissues
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FFPE tissue was cut into 2 μm sections on a microtome and de-paraffinized. After antigen retrieval slides were incubated with primary antibody (the primary antibodies used are listed in Table S3 ) over night at 4 °C and washed with Tris-HCL (Tris hydrochloride) buffer (pH 7.5) followed by a secondary antibody. Antibodies were detected with the Vectastain Elite ABC (avidin-biotin complex) kit (Vector) using DAB (3,3′-diaminobenzidine) (Vector Laboratories and Dako) for brown stainings or AEC (3-Amino-9-ethylcarbazole) (Thermo Fisher Scientific) for magenta stainings. The slides were counterstained with hematoxylin (Vector Laboratories) and mounted with Roti®-Histokitt II (Carl Roth, Germany). Images were captured on an Axioplan2 imaging microscope (Carl Zeiss) equipped with an AxioCamHRc Camera (Carl Zeiss) or slides were scanned with a Vectra Polaris imaging system (PerkinElmer, Hopkinton, MA, USA) and quantified by ImageJ software.
4
Immunohistochemical Analysis of Lung Tissues
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Lung tissues were fixed in 4% formaldehyde and embedded in paraffin. The paraffin section was deparaffinized with xylene and rehydrated with ethanol. For H&E, the slides were stained with H&E (Thermo Scientific, Waltham, MA, USA). In IHC staining, after antigen retrieval, the sections were blocked for endogenous peroxidase activity with 3% H2O2. Bcl-w (Abcam, 1:500 dilution) and HIF-1α (Novus, 1:250 dilution) were incubated overnight at 4°C. After washing with TBS-T (TBS with Tween 20), Vectastain ABC kit and DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA, USA) were used according to manufacturer`s protocols. The signal was detected with cellSens (Olympus, Tokyo, Japan).
5
Immunohistochemical Analysis of CBS in Colon Tissues
6
Immunohistochemistry on Paraffin Sections
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Four μm sections prepared from paraffin blocks were transferred to baths of clearene, then rehydrated though graded alcohol (100%-95%-70%) and immersed in water. Endogenous peroxidase activity was inhibited with incubation of 3% hydrogen peroxide in methanol. Heat-induced epitope retrieval (citrate or EDTA) was performed according to the protocol optimized for the primary antibody to be used. Sections were incubated overnight at 4°C or room temperature with the primary antibody. The following day, the appropriate secondary biotinylated antibody was applied followed by incubation with VECTASTAIN Elite ABC Kit (Vector Laboratories). DAB (3, 3-diaminobenzidine) (Vector Laboratories) was used to reveal specific staining.
7
Quantitative PD-L1 Immunohistochemistry in Mammary Tumors
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The method has been published previously (15 (link)). Briefly, the mammary tumor tissues were fixed with paraformaldehyde (PFA) and embedded in paraffin. Tissue blocks were cut into 5 μm thick sections in a HM 325 rotary microtome (Thermo Scientific). Sections were then deparaffinized and placed in the antigen retrieval solution (Histo-VT One, Nacalai USA). After rinsing thoroughly in 0.1 M PBS, the sections were pre-incubated in a blocking solution and then incubated with mouse PD-L1/B7- H1 antibody (R&D systems). After that, the sections were rinsed and incubated with anti-mouse biotinylated secondary antibody IgG (H+L) (Vector Laboratories). After washing with 0.1 M PBS, the sections were incubated with Avidin/Biotin complex (Vectastain ABC Elite kit; Vector Laboratories) and then chromagen DAB (3′,3′ -diaminobenzidine, Vector Laboratories). The sections were observed under an Olympus CX43 biological microscope. For quantitative assessment of PD-L1 staining, the average optical density (OD) of the regions of interest was measured by Fiji Image J software.
8
Immunohistochemical Staining Protocol
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Immunohistochemical staining was performed as previously (Xu et al., 2016 (link)). Briefly, tissue sections were dewaxed then incubated with 1× target retrieval solution (Dako, Carpinteria, CA, United States) for antigen retrieval, followed by 3% hydrogen peroxide and 5% bovine serum albumin for 30 min, respectively. These sections were incubated with primary antibodies overnight at 4°C. Primary antibodies included anti-TGFβ1 (Santa Cruz Biotechnology, Santa, CA, United States) at 1:100 dilution, anti-HO-1 (Abcam, Cambridge, MA, United States) at 1:100 dilution, anti-cleaved caspase-3 (Abcam, Cambridge, MA, United States) at 1:100 dilution, and anti-Collagen I (Abcam, Cambridge, MA, United States) at 1:100 dilution. Secondary antibodies (1:300–400 dilutions with PBS) were incubated for 1 h in room temperature. Sections were then treated with peroxidase substrate DAB (3, 3-Diaminobenzidine, Vector Laboratories, Burlingame, CA, United States) for coloration and counterstained with hematoxylin.
9
Immunohistochemical Analysis of PAK1 in Testis
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Testis sections of OA patients and recipient mice grafted with human SSCs by PAK1-siRNA 2 or the control siRNA transfection were deparaffinized by xylene twice, hydrated with a series of graded alcohol, and treated with 3% H2O2 (Boster Biological Technology, Guangzhou, China) for 15 min at room temperature to block the endogenous peroxidase activity. After blocking with 5% BSA for 1 hr at room temperature, the sections were incubated with primary antibodies (Table 5 ) at 4°C overnight. After extensive washes with PBS, the sections were incubated with HRP-conjugated second antibody for 1 hr at room temperature. After extensive washing with PBS, DAB (3, 3-diaminobenzidine) (Vector Lab, Burlingame, USA) was used to label PAK1 protein. Finally, the sections were stained with hematoxylin and observed under a Nikon microscope.
10
Immunohistochemical Staining Protocol
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IHC was performed as previously described.3 Briefly, 4‐μm sections of paraffin‐embedded samples were de‐paraffinized, and exposed to appropriate heat‐mediated antigen retrieval. Sections were exposed to primary antibodies at 4°C O/N, and secondary antibodies at RT for 1 h. DAB (3,3′‐diaminobenzidine, Vector Labs) was used to visualize immunoreactivity and Mayer's Hematoxylin (MilliporeSigma) was used to indicate nuclei. The following antibodies were used: PECAM (monoclonal rabbit, 1:300, Millipore), PAI‐1 (polyclonal rabbit, 1:100, Sigma Prestige), SNAI1/2 (polyclonal rabbit, 1:200, Abcam), S100A4 (polyclonal rabbit, 1:200, Sigma Prestige), SMAD4 (monoclonal rabbit, 1:400, Cell Signaling Technology), and secondary antibodies (antirabbit, 1:200, Vector Labs). The Trichrome assay (Abcam) was used according to manufacturer's specifications to evaluate collagen deposition. Slides were imaged using a Zeiss Axio Imager M2 microscope.
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