Sections were deparaffinized, antigens retrieved, and blocked as described above. Primary antibodies used were phospho-AKT (Ser 473) (AF887; 1:200; R&D Systems, Minneapolis, MN) and active β-catenin (clone 8E7, #05-665; 1:300; Millipore, Billerica, MA). After washing, sections were incubated in 3% H2O2 in PBS for 15 minutes to block endogenous peroxidase activity. Sections were then incubated with secondary antibodies for 1 hour at room temperature. Secondary antibodies were Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit (ab6721; 1:1,000; Abcam, Cambridge, MA) and (HRP)-conjugated goat anti-mouse (#7076; 1:1,000; Cell Signaling Technology, Danvers, MA). Protein was visualized using, 3,3' Diaminobenzidine (DAB) substrate kit according to manufacturer’s instructions (Abcam). Sections were counterstained with Hematoxylin, dehydrated, and mounted with Cytoseal60 (Thermo Scientific). Cells were visualized using Nikon Eclipse E400 (Tokyo, Japan), and images were captured at 40× or 100× total magnification using QCapture software (Surrey, BC, Canada).
3 3 diaminobenzidine dab substrate kit
Manufactured by Abcam
Sourced in United States
The 3,3' Diaminobenzidine (DAB) substrate kit is a reagent used in immunohistochemistry and immunocytochemistry applications. It serves as a chromogenic substrate for the detection of target proteins or antigens in tissue sections or cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Cytoseal 60, by Thermo Fisher Scientific (1 mentions)
Af887, by R&D Systems (1 mentions)
Eclipse e400, by Nikon (1 mentions)
Hematoxylin, by Thermo Fisher Scientific (1 mentions)
Active β catenin clone 8e7, by Merck Group (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
Mouse anti α sma, by Abcam (1 mentions)
Goat serum, by Beyotime (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Mayer s hematoxylin, by ScyTek Laboratories (1 mentions)
Xylene, by Merck Group (1 mentions)
Aperio digital pathology slide scanner, by Leica (1 mentions)
Envision dual link system hrp dab kit, by Agilent Technologies (1 mentions)
4 protocols using 3 3 diaminobenzidine dab substrate kit
1
Immunohistochemical Evaluation of Phospho-AKT and Active β-Catenin
2
Immunohistochemical Analysis of Liver Tissue
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For immunohistochemistry analysis, liver sections were de-paraffinized in two changes of xylene for 15 minutes each and then hydrated through a graded series of ethanol (100, 95, 70, 50%) and deionized water, each for 5 minutes. The tissue sections were processed for heat-mediated antigen-retrieval using Tris-EDTA or citric acid buffer for 15 minutes (ZSGB-BIO, Beijing), and then cooled at room temperature. Sections were blocked for 1 hr at room temperature in PBS with 10% goat serum (blocking buffer), and then incubated at 4°C overnight with primary antibodies in blocking buffer (anti-human CD4, 1:500; anti-human CD8, 1:1000; anti-mouse CD4, 1:1000; anti-mouse CD8, 1:500; anti-mouse α-Sma, 1:1000, Abcam, Cambridge, UK). The next day, sections were washed three times with PBS for 5 min, and incubated for 2 hrs at room temperature with secondary antibody (goat anti-rabbit F(ab’)2-HRP). Staining was developed using 3, 3’ Diaminobenzidine (DAB) substrate kit (Abcam) according to the manufacturer’s instructions. Staining was then assessed at × 100 or × 200 magnification in a total of 3-5 fields/section/sample, using cellSens Dimension software (Olympus) for computerized quantification and results were expressed as the intensity of positive staining per field.
3
Histopathological Analysis of Liver Fibrosis
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To explore histopathologic changes, the liver tissues were fixed with 10% neutral formalin, embedded in paraffin, and then sectioned transversely (5-μm-thick). The thin liver tissue sections were then stained with hematoxylin and eosin (H&E). The deposition of collagen fibers in hepatic sections was measured using Masson staining and Sirius red staining by the conventional protocols. Fibrosis stage was assessed according to the Ishak score [31 (link)]. As for IHC staining, embedded liver sections were dewaxed, and antigens were retrieved via sodium citrate heating. Endogenous peroxidase was removed by adding 30% H2O2, and an immunohistochemical pen was used to draw a circle around the tissue. Then, 5% goat serum (#C0265, Beyotime Biotechnology) was added to block the liver tissues. The liver sections were then incubated with primary antibodies (Supplementary Table 1 ) at 4 °C overnight. Sections were then washed, followed by incubation with secondary antibodies (Supplementary Table 1 ) for 1 h at room temperature. IHC staining was observed using 3,3′-diaminobenzidine (DAB) substrate kit (#ab64238, Abcam, USA) and were counterstained with hematoxylin. All images were captured under a microscope. The percentage of collagen areas or positive signal expression fields was measured by Image Pro Plus software (Media Cybernetics, USA).
4
Immunohistochemical Analysis of Tumor Markers
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The paraffin-embedded tissue sections were heated at 60 °C for 1 h, and dewaxed in xylene (Sigma-Aldrich, St. Louis, MO, USA). The tissue sections were rehydrated in graded ethanol from 95% to 75%, and finally in phosphate buffered solution with 0.05% Tween-20 (PBST). The tissue slides were heated in 10 mM citric acid buffer with 0.05% Tween-20 (pH = 6.0) at 121 °C for 3 min using a pressure cooker for antigen retrieval, and the tissue sections were incubated with peroxidase blocking reagent [RTU, EnVisionTM +Dual Link System-HRP (DAB+) kit, Code K4065, Dako, CA, USA] for 5 min, then blocked with goat serum for 30 min. After blocking, the tissue sections were incubated with antibodies against cyclin D1 (cat. no. GTX108624), PD-1 (cat. no. GTX128435, GeneTex, Irvine, CA, USA), 18hosphor-AKT (cat. no. 4060, Cell Signaling Technology, Beverly, MA, USA), and VEGF (ABS82, Millipore, Bedford, MA, USA) at 4 °C overnight, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. After being rinsed with PBST, the sections were developed in 3′,3′-diaminobenzidine (DAB) substrate kit (Abcam, ab64238), and counterstained with Mayer’s hematoxylin (ScyTek Laboratories, UT, USA). All sections were scanned by an Aperio digital pathology slide scanner (Leica Biosystems, Buffalo Grove, IL, USA).
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