Cells (60,000 cells/well) were seeded in 48-well plates for overnight cultivation. The cells were fixed with 4% paraformaldehyde-containing phosphate-buffered saline (PBS) for 30 min at room temperature. Cells were incubated in 0.2% (v/v) Triton X-100 solution to change their membrane permeability, followed by incubation with PBS containing 0.3% (v/v) hydrogen peroxide (30 min). Nonspecific binding was blocked using 3% (w/v) bovine serum albumin (BSA) in PBS for 30 min. Cells were incubated with 3 µg/mL of rabbit anti-EBF1 polyclonal antibody (Abcam, MA, USA) at room temperature for overnight, followed by treatment with peroxidase-conjugated EnvisionTM secondary antibody (DAKO, Glostrup, Denmark). The color was developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate kit (Vector Laboratories, Inc., CA, USA) and washed with distilled water. The stained cells were dehydrated with ethanol and air dried for overnight. The stained cells were analyzed using an inverted microscope. The semi-quantitative analysis was calculated in ImageJ Fiji software using the method described by Crowe and Yue 33 .
3 3 diaminobenzidine tetrahydrochloride dab substrate kit
Manufactured by Vector Laboratories
Sourced in United States
3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate kit is a laboratory product used for immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) applications. The kit provides a chromogenic substrate for the detection of peroxidase enzyme activity.
Automatically generated - may contain errors
4 protocols using 3 3 diaminobenzidine tetrahydrochloride dab substrate kit
1
Immunocytochemical Analysis of EBF1 Expression
2
Quantifying Phosphorylated Tau in Neurons
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Paraffin-embedded tissue sections were deparaffinized followed by incubation with hydrogen peroxide (0.3%) for 20 min. Sections were then incubated with anti-p-Tau Ser214 antibody (1:100, Thermo Fisher Scientific, IL, USA; catalogue number: 44-742G) overnight at 4 °C. The samples were rinsed with PBS and incubated for 20 min with secondary antibody HRP Envision kit (Dako, CA, USA). Following another PBS wash, sections were incubated for 15 min with 3, 3′-diaminobenzidine tetrahydrochloride (DAB Substrate Kit, Vector Laboratories Inc., CA, USA). They were rewashed with PBS, counterstained with hematoxylin, dehydrated, cleared in xylene, and cover slipped for light microscopic investigation. Quantification of p-Tau in pyramidal neurons was performed by measuring the optical density from four random fields in each section and averaged using the image analysis software (Image J, version 1.46a, NIH, MD, USA) (Abdelkader et al. 2022 (link)).
3
Immunohistochemistry and TUNEL Assay Protocol
4
Immunohistochemical Detection of MMR Proteins
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TMAs were performed to detect MMR proteins using the IHC technique. The TMA samples were deparaffinized and rehydrated in xylene and ethanol (100%, 90%, 80% and 70%), respectively. Retrieval was then preformed in 1xTris-EDTA using a pressure cooker (MLH1 and MSH6) and autoclave (MSH2 and PMS2). Endogenous peroxidase activity and non-specific binding were performed using 0.3% (v/v) of hydrogen peroxide (H2O2) and 10% skim milk, for 10 min for each step. Primary antibodies were then added and incubated at 4 °C overnight. After washing with phosphate buffer saline (PBS) plus 0.1% Tween 20, horseradish peroxidase (HRP)-conjugated secondary antibodies were added and incubated for 1 h. After washing, a 3,3′ diaminobenzidine tetrahydrochloride (DAB) substrate kit (Vector Laboratories, Inc., CA) was used to develop the signal. Mayer’s hematoxylin was used for the counterstain. Dehydration was performed with 70%, 80%, 90% and 100% ethanol and xylene, respectively. After mounting, tissue sections were observed under a light microscope. The status of MMR proteins was evaluated by pathologists when the positive control exhibited a signal after staining. The positive control was composed of internal and external positive controls. Internal positive controls included adjacent normal liver tissues, stomal cells and lymphocytes, while the external control was normal liver tissue.
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