TUNEL assay was performed to detect cell apoptosis using the In Situ Cell Apoptosis Detection Kit (Boster, China). Microglia were inoculated on coverslips that had been coated with PLL and treated with reagents. After 24 h, microglia were washed three times in PBS and fixed with 4% PFA for 30 min at 37°C. After washing three times with PBS, microglia were digested using Proteinase K (1:200 diluted in 0.01M TBS) at room temperature for 30 s and washed three times in 0.01M TBS. According to the manufacturer’s instructions, microglia were incubated with TUNEL reaction reagent, biotin-labeled anti-digoxin antibody, and SABC diluent. Microglia were then incubated with DAPI for nuclear staining, and finally mounted to the slides. Apoptotic cells were imaged by a fluorescence microscope.
In situ cell apoptosis detection kit
Manufactured by Boster Bio
Sourced in China
The In Situ Cell Apoptosis Detection Kit is a laboratory equipment used to detect and analyze apoptosis, a form of programmed cell death, in cells. The kit provides the necessary reagents and protocols to perform this analysis.
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7 protocols using in situ cell apoptosis detection kit
1
Apoptosis Detection in Microglia
2
TUNEL Staining for Apoptosis Detection
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Paraffin sectioning was performed at 3.5 μM. After dewaxing in xylene and rehydration in gradient ethanol, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling staining (TUNEL staining) was conducted with an in situ cell apoptosis detection kit (Boster, China, Cat# MK1020). The above staining procedures were done according to the manufacturer’s instructions. The number of TUNEL-positive cells was counted in 10 random high-power fields.
3
Apoptosis Detection in Tumor Tissues
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Oridonin was found to kill malignant cells by inducing cellular apoptosis (13 (link)). TUNEL, as first described by Gavrieli (14 (link)), is an assay for localization of apoptotic DNA fragmentation in situ. Therefore, fresh-frozen sections of tumor tissues were tested by TUNEL staining according to the manufacturer’s instructions. In situ cell apoptosis detection kit was purchased from Boster Biological Technology (Wuhan, Hubei, China). The results of the bluish-violet were regarded as an indicator of apoptotic cells. TUNEL-positive nuclei, a pyknotic nucleus with bluish-violet granules, were visualized and analyzed under a light microscope (SZ61; Olympus Corporation, 40/400×). Cell numbers were counted in 5 random fields and apoptosis indexes (AI) were calculated by the following equation as: AI = positive cell number/total tumor cell number. All the slides were examined independently by two pathologists, and their findings were in concordance.
4
Detecting Apoptosis in Liver IRI
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Apoptotic cells in IRI liver tissue were detected by terminal deoxynucleotidyl transferase–mediated uridine triphosphate nick-end labeling assays using the In Situ Cell Apoptosis Detection Kit (Boster, China), as described previously13 (link).
5
Quantifying Myocardial Apoptosis and Proliferation
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The number of apoptotic cells in the ischemic zone was measured by the TUNEL assay using an in situ cell apoptosis detection kit (Boster) according to the manufacturer's instructions. The microscopic ten areas were recorded and the TUNEL-positive cells were calculated by Image-Pro Plus 6.0.
For immunohistochemistry, the tissue sections were incubated with diluted primary antibodies against rat Ki67 (1 : 200) (Santa Crus) after blocking the nonspecific areas with 5% bovine serum albumin. Then, the tissue sections were incubated with biotin-conjugated anti-rabbit immunoglobulin G and streptavidin biotin and colored with 3,3′-diaminobenzidine. Finally the myocardial tissues in the border zone were photographed after staining with haematin. The cells positive for Ki67 were counted by Image-Pro Plus 6.0.
The myocardial tissue below the ligation position was fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 μm serial sections which were then used for Masson's trichrome staining. Images were captured using a stereo microscope (SMZ-168, Motic, China) and were analyzed using Image-Pro Plus 6.0.
For immunohistochemistry, the tissue sections were incubated with diluted primary antibodies against rat Ki67 (1 : 200) (Santa Crus) after blocking the nonspecific areas with 5% bovine serum albumin. Then, the tissue sections were incubated with biotin-conjugated anti-rabbit immunoglobulin G and streptavidin biotin and colored with 3,3′-diaminobenzidine. Finally the myocardial tissues in the border zone were photographed after staining with haematin. The cells positive for Ki67 were counted by Image-Pro Plus 6.0.
The myocardial tissue below the ligation position was fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 μm serial sections which were then used for Masson's trichrome staining. Images were captured using a stereo microscope (SMZ-168, Motic, China) and were analyzed using Image-Pro Plus 6.0.
6
TUNEL Staining for Apoptosis Analysis
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Apoptosis analysis was performed by TUNEL (terminal deoxynucleotide transferasemediated dUTP nick end-labeling) staining using an in situ cell apoptosis detection kit (Boster Biological Technology, Wuhan, Hubei, China) according to the manufacturer's instructions. The results of the bluish-violet were taken as an indicator of apoptotic cells. TU-NEL-positive nuclei, a pyknotic nucleus with bluish-violet granules, were visualized and analyzed under a light microscope (200×). Cell numbers were counted in 5 random fields and apoptosis indexes (AI) were calculated as the ratio of the positive cell number to the total tumor cell number, based on the mean value from five high power fields via computer--assisted assay.
7
Apoptosis Detection Using TUNEL Assay
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Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed using an In Situ Cell Apoptosis Detection Kit (Boster, Wuhan, China), according to the instructions of the manufacturer and a previous description (2011) . Five fields of each slice were photographed for analyzing the average optical density (AOD) in each field.
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