Glioma tissues and adjacent normal tissues were made into paraffin sections, dewaxed with xylene, and hydrated with 95% ethanol. Then, the sections were repaired by immersion of endogenous peroxidase at 37.0°C in citric acid buffer for 30 min, blocked with 5% goat serum at room temperature for 60 minutes, and incubated with antibodies against LAIR‐1 (1:4000, 67200‐1‐Ig, Proteintech). Next, the horseradish peroxidase (HRP)‐conjugated secondary antibody was applied at 37°C for 45 minutes and diaminobenzidine (DAB) solution (Beyotime) was used to stain the slice, and we counterstained the nuclei with hematoxylin (Beyotime). Immunohistochemical staining was independently evaluated by two pathologists without prior knowledge of patient characteristics. According to the degree of positive staining (antigen content), it can be divided into: Weak positive (+); Moderate positive (++); Strong positive (+++). According to the number of positive cells, they can be divided into: Weakly positive (+, <25%); Medium positive (++, 25%–49%); Strong positive (+++, >50%).
Diaminobenzidine dab solution
Manufactured by Beyotime
Diaminobenzidine (DAB) solution is a chromogenic substrate used in various immunohistochemical and histochemical applications. It is commonly used to visualize the presence and localization of specific target proteins or enzymes in biological samples. When exposed to the appropriate enzyme, the DAB solution undergoes a reaction that produces a brown-colored precipitate, enabling the detection and identification of the target molecules.
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Lab products found in correlation
Hematoxylin, by Beyotime (1 mentions)
Anti c jun antibody, by Abcam (1 mentions)
Anti c fos antibody, by Abcam (1 mentions)
Anti atf2 antibody, by Abcam (1 mentions)
Citrate antigen retrieval solution, by Beyotime (1 mentions)
Hematoxylin staining solution, by Beyotime (1 mentions)
Ab15580, by Abcam (1 mentions)
3 protocols using diaminobenzidine dab solution
1
Immunohistochemical Analysis of LAIR‐1 in Glioma
2
Immunohistochemical Evaluation of Transcription Factors in Lung and Colon
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IHC assay was carried out to assess activating transcription factor 2 (ATF2), c-jun, and c-fos expressions in lung and colon tissues. Briefly, 6 µm lung or colon sections were de-paraffinized in xylene solution and hydrated in a decreasing gradient ethanol solution. Sections were then treated with citrate antigen retrieval solution (Beyotime Biotechnology, 20 min, 95 ℃), permeabilized using 0.5% Triton X-100 solution (20 min), incubated with 3% H2O2 solution, and coated with 3% bovine saline albumin (BSA, Beyotime Biotechnology). Sections were then treated with anti-ATF2 antibody (1:250), anti-c-jun antibody (1:250), and anti-c-fos antibody (1:250, Abcam Biotechnology, MA, USA) overnight at 4 ℃ and HRP-conjugated secondary antibody for 60 min. Relative expressions were visualized using a diaminobenzidine (DAB) solution (Beyotime Biotechnology) and the cell nucleus was stained using hematoxylin solution. Results were observed under a microscope (Nikon, Japan).
3
Immunohistochemistry Protocol for Ki-67 Analysis
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Tumor tissues were xed in 10% formalin and embedded in para n. Specimens were sectioned at 5-µm thickness. The sections were stained using routine immunohistochemical techniques. Brie y, after pretreatment with heat-mediated antigen retrieval for 20 min in a microwave oven, the endogenous peroxidase activity was blocked by incubating the tissues in 3% H 2 O 2 for 30 min. After blocking for 15 min with 10% normal goat serum, the sections were treated with primary antibodies against Ki67 (1:500 dilution; overnight exposure) (ab15580, Abcam). HRP-conjugated secondary antibodies were used for detection. Next, the sections were exposed to Diaminobenzidine (DAB) solution (P0203, Beyotime Biotechnology), and counterstained by Hematoxylin Staining Solution (C0107, Beyotime Biotechnology). Finally, tumor specimens were observed under the microscope. The substitution of PBS for primary antibody was used as negative control. For Ki-67 index, numbers of positively stained cells were expressed as a percentage of the total number of cells examined.
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