Terminal transferase-mediated biotin 2′-deoxyuridine, 5′-triphosphate nick end labeling (TUNEL) assay was performed to evaluate apoptosis of cells with a TUNEL assay kit (ab206386, Abcam, Cambridge, UK) as previously described (27 (link)). The hippocampus tissue was first deparaffinized and treated with 20 μg/ml deoxyribonuclease-free proteinase K (AM2548, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 20 min and was then washed with PBS three times (5 min for each). After being fixed at room temperature for 15 min, the samples were washed with PBS for another three times (5 min each time). Next, each sample was added with 50 μl of biotin labeling and incubated for 1 h at 37°C, followed by processing with a 3,3′-diaminobenzidine kit (DA1015, Solarbio, Beijing, China) and PBS washing. Then, the samples were sealed after clearing with methylbenzene (244511, Sigma-Aldrich, USA) for 5 min. The apoptotic cells were observed using a stereomicroscope (SZX10, Olympus, Japan) under × 100 and × 200 magnifications.
3 3 diaminobenzidine kit
Manufactured by Solarbio
Sourced in China
The 3,3'-diaminobenzidine kit is a laboratory reagent used for the detection and visualization of certain proteins or enzymes in biological samples. It serves as a chromogenic substrate, producing a brown-colored precipitate upon enzymatic reaction, enabling the identification and localization of target molecules.
Automatically generated - may contain errors
2 protocols using 3 3 diaminobenzidine kit
1
TUNEL Assay for Apoptosis Analysis
2
Evaluation of NF-κB and Cell Proliferation in MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSCs were harvested and lysed in RIPA buffer (Sigma-Aldrich; Merck KGaA; cat. no. 20-188) at 4°C. Following centrifugation (14,000 × g for 15 min at 4°C), the protein concentration was determined using the Bradford method (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer's protocol. A total of 20 µg total protein sample was separated by 10% SDS-PAGE (Hangzhou Fude Biological Technology Co., Ltd., Hangzhou, China) and transferred onto a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% nonfat milk for 1 h at room temperature then incubated with antibodies against p65 NF-κB (Abcam; cat. no. ab16502; 0.5 µg/ml), PCNA (Abcam; cat. no. ab18197; 1 µg/ml) and Fas (Abcam; cat. no. ab82419; 1:1,000) at 4°C overnight. The nitrocellulose membrane was then incubated with anti-rabbit IgG (Abcam; cat. no. ab191866; 1:500) secondary antibodies, for 1 h at room temperature. Finally, protein was detected using a 3,3′-diaminobenzidine kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). GAPDH served as the internal control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!