Formalin-fixed paraffin-embedded skin sections were deparaffinized, rehydrated and then TUNEL stained as described before [35 (link)], with modification. The sections were incubated in 0.85% NaCl and 0.01 M phosphate buffered saline (PBS) for 5 minutes each, fixed in 4% paraformaldehyde in PBS for 15 minutes and then washed with PBS. The sections were then incubated with proteinase K (20 μg/ml) for 10 minutes, washed with PBS, blocked with avidin/biotin solutions (Invitrogen, USA) for 15 minutes each and incubated in a TUNEL reaction buffer (30 mM Tris-HCl, pH 7.2; 140 mM sodium cacodylate; and 1 mM CoCl2). Subsequently, the sections were incubated in a humidified chamber for 90 minutes at 37°C with TUNEL reaction mixture-containing biotin-16-dUTP (10 nmol/ml) (Roche, USA) and terminal deoxynucleotide transferase (TdT) (200 U/ml) (Roche). Negative controls were incubated without TdT. Sections were then washed in PBS, incubated with ABC reagents (Vectastain ABC Elite Kit; Vector Laboratories, USA) for 60 minutes and stained with 3, 3 –diaminobenzidine (DAB) substrate (Dako, USA).
3 3 diaminobenzidine dab substrate
Manufactured by Agilent Technologies
Sourced in Denmark, United States
3,3'-diaminobenzidine (DAB) substrate is a chromogenic substrate used in immunohistochemistry and immunocytochemistry applications. It produces a brown-colored precipitate at the site of enzyme activity, allowing for the visualization and detection of target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
Anti ki67, by Thermo Fisher Scientific (2 mentions)
Anti caspase 3, by Biocare Medical (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Bx60 fluorescence microscope, by Olympus (1 mentions)
Glial fibrillary acidic protein (gfap), by Novus Biologicals (1 mentions)
Anti gli1, by Santa Cruz Biotechnology (1 mentions)
Anti ptch1, by Santa Cruz Biotechnology (1 mentions)
Anti cd31, by Dianova (1 mentions)
Anti pimonidazole, by Hypoxyprobe (1 mentions)
α sma, by Novus Biologicals (1 mentions)
Proteinase k solution, by Merck Group (1 mentions)
Pdgfrβ, by Cell Signaling Technology (1 mentions)
Cxcl10, by Abcam (1 mentions)
Streptavidin peroxidase, by Vector Laboratories (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti phospho c met, by Cell Signaling Technology (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Tissue studio, by Definiens (1 mentions)
Anti c met, by Cell Signaling Technology (1 mentions)
15 protocols using 3 3 diaminobenzidine dab substrate
1
TUNEL Staining of Formalin-Fixed Paraffin-Embedded Skin
2
Immunohistochemical Analysis of Prostate Tissue
3
Localization of Microglia and Astrocytes in Hippocampus
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For the localization of microglia and astrocytes in the hippocampus, immunohistochemistry was performed. Also, after an antigen retrieval step using citrate buffer (pH 6.0) at 96 °C for 30 min, endogenous peroxide activity was blocked, and the hippocampal sections were incubated overnight (~ 16 h) at 4 °C with the rabbit polyclonal antibody anti-Iba1 or anti-glial fibrillary acidic protein (GFAP; Novus Biological, Littleton, CO, USA, Cat No. NBP2-16908 and NB300-141), which are microglia and astrocyte markers, respectively, and are 1:1000 diluted in 2% BSA. After washing, the sections were incubated with a secondary biotinylated antibody (LAB-SA Detection kit, Invitrogen, Paisley, UK, Cat No. 95-9843). Positive staining was detected using a peroxidase-conjugated streptavidin complex, and colour was developed using 3,3′-Diaminobenzidine (DAB) substrate (Dako, Cambridge, UK, Cat No. K3468). Lastly, the sections were counterstained with haematoxylin.
4
Immunohistochemical Analysis of Tumor Samples
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After antigen retrieval and blocking (Supplemental Table S1 ), tumor sections were incubated overnight at 4°C with anti-pimonidazole (1/400, Hypoxyprobe, Massachusetts, USA), anti-caspase-3 (ready to use, Biocare Medical, Concord CA, USA) or anti-CD31 (1/25, Dianova, Hamburg, Germany), anti-GLI1 (1/50, Santa-Cruz), anti-GLI2 (1/1000, Rockland), anti-PTCH1 (1/300, Santa Cruz); or for 30 min at room temperature with anti-Ki67 (ready to use, Thermo Scientific). Appropriate secondary antibodies followed by 3.3′-diaminobenzidine (DAB) substrate (DAKO, Glostrup, Denmark) were used to visualize antigen presence.
Quantification of Ki67 was performed by counting the number of Ki67 positive nuclei in the tumor tissue. Mean vessel density (MVD) was assessed as the number of blood vessels (CD31+) per field for 10 high-power fields per tissue specimen. Tumor hypoxic fraction was determined as the percentage of cytoplasmic pimonidazole positive cells. The apoptotic fraction was determined by assessing the number of caspase-3 positive cells per tissue section.
Quantification of Ki67 was performed by counting the number of Ki67 positive nuclei in the tumor tissue. Mean vessel density (MVD) was assessed as the number of blood vessels (CD31+) per field for 10 high-power fields per tissue specimen. Tumor hypoxic fraction was determined as the percentage of cytoplasmic pimonidazole positive cells. The apoptotic fraction was determined by assessing the number of caspase-3 positive cells per tissue section.
5
Immunohistochemical Analysis of Tissue Samples
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For immunohistochemical analysis on the retrieved histological sections, endogenous peroxidase was blocked with 3% hydrogen peroxide, while nonspecific antibody binding o was inhibited with 10% normal goat serum for 30 min. Primary antibodies (Table 1 ) were diluted 1:500 and incubated for 2 h. After washing, samples were incubated with secondary antibody (Polink-1 HRP Broad Spectrum AEC/DAB Detection Kit, 1:400 dilution; GBI Labs Inc., Bothell, DC, USA) for 40 min, and incubated with Horse Radish Peroxidase-conjugated 3-amino-9-ethylcarbazole (AEC) substrate (Dako, Glostrup, Denmark) for 5–7 min or 3,3′-diaminobenzidine (DAB) substrate (Dako) for 1–2 min. The stained sections were mounted with distyrene plasticizer-xylene (DPX) Mounting Solution, and photo-documented with bright field microscopy.
6
Hippocampal Expression of PPAR-γ and BDNF
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Hippocampal slices were deparaffinized and rehydrated as described above. After antigen retrieval, slices were incubated with 3% H2O2 for 15 min and blocked in goat serum (containing 0.1% Triton X-100) for 30 min followed by incubation overnight at 4°C with primary antibodies to PPARγ (1 : 200) and BDNF (1 : 200). Then, the slices were washed three times with PBS and incubated with the horseradish peroxidase (HRP) conjugated goat anti-rabbit or anti-mouse IgG (1 : 100) secondary antibody for 2 h at room temperature followed by incubation with 50 μL 3,3′-diaminobenzidine (DAB) substrate (DAKO, Denmark) at room temperature for 10 min. The number of immunoreactive cells in the hippocampus was assessed using light microscopy (DP70; Olympus, Japan). At least three different fields (200 × 200 μm) per slice were randomly selected for visualization. The mean optical density in the hippocampus region was calculated and used to determine PPARγ and BDNF expression levels.
7
Immunohistochemical Analysis of Skin Tissue
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For single antibody staining, formalin-fixed, paraffin-embedded 5-μm skin tissue sections were de-paraffinized and rehydrated through a graded series of ethanol. Antigens were retrieved by incubation with a proteinase K solution (EMD Millipore, Billerica, MA, USA) for 5 minutes. Blocking was done by 2.5% normal horse serum for 1 h. Tissue sections were then incubated with primary antibody to αSMA (1:100, Novus Biologicals, Littleton, CO, USA), PDGFRβ (1:50, Cell Signaling Technology, Danvers, MA, USA), rat anti-mouse CD45 Ab (1:100, BD Pharmingen, San Diego, CA, USA), phospho-Smad2 (Ser465/467) Ab (1:100, Cell Signaling), or vWF (1:1000, DAKO, Santa Clara, CA, USA) at 4 °C for 16 h. Next, sections were incubated with ImmPress horseradish peroxidase (HRP) anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for 30 minutes. The color was developed using 3,3-diaminobenzidine (DAB) substrate (DAKO). Immunohistochemical images were collected using a microscope (BH-2; Olympus, Center Valley, PA, USA).
8
Immunohistochemical Analysis of CCL5 and CXCL10
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Formalin-fixed paraffin-embedded sections (3 μm) were de-paraffinized in xylene, rehydrated through graded alcohol, and washed briefly in tap water. Endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide for 30 min. To retrieve antigenicity, sections were boiled in 10 mM citrate buffer (pH 5.8) for 30 min in a microwave oven (800 W). Next, sections were incubated with goat serum diluted in PBS (pH 7.4) for 30 min at room temperature. Subsequently, sections were incubated at 4°C overnight with the primary antibodies specific for CCL5 (dilution 1: 200) or CXCL10 (dilution 1:200) (Abcam, Cambridge, UK). Then, sections were rinsed in fresh PBS and incubated with horseradish peroxidase-linked secondary antibodies at room temperature for 60 min. Finally, sections were stained with 3, 3′-diaminobenzidine (DAB) substrate (Dako, Carpinteria, CA, USA) and counterstained with Mayer's hematoxylin. Non-immune rabbit IgG at the same dilution was used as negative control. Photos were recorded under a microscope (Leica, Wetzlar, Germany).
9
Immunohistochemical Detection of CCL2
10
Immunohistochemical Analysis of Mouse Tumor Tissues
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Immunohistochemistry on paraformaldehyde fixed, paraffin embedded, 3 μm thick sections of mouse tumor tissues was performed manually using anti-Ki67 (1:2000), anti-CD31 (1:200) (abcam), anti-cleaved Caspase3 (1:1000), anti-phospho cMET (1:1000), anti-cMET (1:1000), anti-MMP9 (1:1000) (Cell Signaling Technologies), anti-E-cadherin (1:1000) (BD Biosciences) and anti-ILEI (1:1000) [6 (link)] primary antibodies and Lab Vision™ UltraVision™ LP Detection System (Thermo Scientific) with 3,3′-diaminobenzidine (DAB) substrate (Dako) for detection according to the manufacturer’s instruction. Cell nuclei were visualized by hematoxylin staining. Histological samples were scanned using a Pannoramic MIDI slide scanner (3D Histech) with a 40X objective. Subsequently, quantification of immunomhistochemistry was performed by the histomorphometric software package Tissue Studio® (Definiens AG). The E-cadherin membrane score was obtained by the formula: 3 x ratio of high membrane staining intensity + 2 x ratio of medium membrane staining intensity +1x ratio of low membrane staining intensity, giving a range of 1 to 3.
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