TUNEL staining was performed using the POD in situ cell death detection kit (Roche, USA). Briefly, sections were deparaffinized in xylene, rehydrated in decreasing concentration of ethanol, and then treated by nuclease free proteinase K for 15 min at room temperature before endogenous peroxidase was blocked in 3% H2O2 in methanol. Terminal deoxynucleotidyl transferase (TdT) in reaction buffer was applied to sections for 1 h at 37°C. Following washes, the slides were covered by converter-POD solution for 30 min at 37°C. Apoptotic cells were detected after incubation in 3,3’-diaminobenzidine (DAB) chromogen (Beyotime Biotechnology, China) for approximately 8 min, and the slides were counterstained with hematoxylin.
3 3 diaminobenzidine dab chromogen
Manufactured by Beyotime
Sourced in China
3,3'-diaminobenzidine (DAB) chromogen is a chemical compound used as a chromogenic substrate in various biochemical and immunohistochemical applications. It is a brown, crystalline powder that undergoes an enzymatic reaction, resulting in the formation of a brown precipitate, which can be detected and visualized under a microscope.
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3 protocols using 3 3 diaminobenzidine dab chromogen
1
TUNEL Staining for Apoptosis Detection
2
TUNEL Staining for Apoptosis Detection
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TUNEL staining was performed using the POD in Situ Cell Death Detection kit (Roche, USA). Sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, then treated by nuclease free Proteinase K for 15 min at room temperature before endogenous peroxidase was blocked in 3% H2O2 in methanol. Terminal deoxynucleotidyl transferase (TdT) in reaction buffer was applied to sections for 1h at 37°C. Following washes, the slides were covered by converter-POD solution for 30 min at 37°C. Apoptotic cells were detected after incubation in the 3, 3′-diaminobenzidine (DAB) chromogen (Beyotime Biotechnology) for approximately 8 min and slides were counterstained with hematoxylin.
3
Immunohistochemical Analysis of NF-κB and Notch1
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The tissue samples underwent fixation with 4% PFA for 48 hours, followed by decalcification using a 10% EDTA solution for 20 days, and then were prepared into 5 µm sections. NF-κB p65 (AN365, 1:500, Beyotime, Shanghai, China), Notch1 (AF5249, 1:200, Beyotime, Shanghai, China) antibody was added and incubated for 60 minutes, followed by rinsing with distilled water and placement in PBS. Goat anti-rabbit IgG-HRP polymer (ab150077, 1:1000, abcam, Cambridge, UK) was added and incubated for 40 minutes, followed by rinsing with distilled water and placement in PBS. 3,3 ′ -Diaminobenzidine (DAB) chromogen (P0202 Beyotime, Shanghai, China) was applied for 3 minutes, and the reaction was controlled under a microscope, terminated by rinsing with tap water. After rinsing with distilled water, counterstaining was performed, and the slides were coverslipped. The presence of yellow or brown particles in the cytoplasm and/or nucleus was considered as positive cells.
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