To determine apoptosis in VSC4.1 neurons following OGD/R injury, a TUNEL assay was performed with the Apoptosis Detection Kit (Boster, Wuhan, China). The assay was carried out completely according to the manufacturer's protocol. TUNEL was visualized with DAB staining. The apoptotic neurons were those with either tightly clustered brown staining or more diffuse brown TUNEL staining confined within the cell. With the microscope under a 20x objective, at least 1000 neurons from six random fields in each group were chosen to quantify the total of the TUNEL-positive cells.
Apoptosis detection kit
Manufactured by Boster Bio
Sourced in China
The Apoptosis detection kit is a laboratory tool designed to detect and analyze programmed cell death (apoptosis) in various biological samples. It provides the necessary reagents and protocols to perform standard apoptosis assays.
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5 protocols using apoptosis detection kit
1
Quantifying Apoptosis in OGD/R-Injured Neurons
2
Apoptosis Quantification in Cardiac Cells
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A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was used to assess the apoptosis with an apoptosis detection kit (Boster Biological Technology, Ltd., Wuhan, China) according to the manufacturer’s instructions. Passage 3 CDCs (5×103) were seeded onto a coverslip and cultured with or without isoproterenol (10−6 M) containing medium for 72 h. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min and incubated with cold (2–8 ℃) 0.5%TritonX-100 for 2 min. The cells were incubated with 50 µL of TdT enzyme working solution at 37 ℃ for 60 min in the dark. After washing with PBS for 5 min ×3 times, 50 µL of Streptavidin-HRP working solution was added to each slide at 37 ℃ for 60 min in the dark. The slide was incubated with 50~100 µL DAB working solution at room temperature for 10 min. The slide was visualized under a microscope (BX51, OLYMPUS, Japan). Positively stained cell nuclei were dark brown.
3
Apoptosis Analysis in Murine Spleen
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At 0 h (uninfected), 12 h, 24 h, and 72 h postinfection, eight mice in each group were sacrificed, the spleen tissues were analyzed and photographed. Spleen tissues were fixed in 4% paraformaldehyde, and processed routinely in paraffin. Then, they were dehydrated, embedded in paraffin, sectioned (thickness, 4~5 μm) and stained with hematoxylin and eosin (H&E) or TUNEL assay. The DNA fragmentation indicative of apoptosis was tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay14 . Slides were stained by TUNEL using an apoptosis detection kit according to manufacturer ((Boster, Wuhan, China) instructions. Slides were viewed under a light microscope (BX 43; Olympus, Tokyo, Japan) and photographed.
4
TUNEL Assay for Apoptosis Detection
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Five-micrometer-thick tissue sections were used for TUNEL staining with the apoptosis detection kit (Boster Biological Technology Co., Ltd., Wuhan, China). Briefly, the slides were deparaffinized, rehydrated, and treated with proteinase K (20 mg/ml) for 15 min at room temperature. The slide was preliminarily incubated with labeling buffer (digoxigenin-dUTP) at room temperature for 2 h. Then blocking solution incubated 30 min at room temperature, and added the reaction mixture containing anti-digoxigenin antibody and Fluorescein-Streptavidin (FITC, green), incubated at 37°C for 30 min. DAPI was used for nuclear counterstaining.
The HK-2 cells (1 × 106 cells/well) were seeded on six-well chamber slides. After different treatments, the slides were detected with the apoptosis detection kit (CY3, red). DAPI was used for nuclear counterstaining. TUNEL-positive cells were imaged under a laser confocal microscope (Nikon, Ti-E and A1 plus).
The HK-2 cells (1 × 106 cells/well) were seeded on six-well chamber slides. After different treatments, the slides were detected with the apoptosis detection kit (CY3, red). DAPI was used for nuclear counterstaining. TUNEL-positive cells were imaged under a laser confocal microscope (Nikon, Ti-E and A1 plus).
5
Apoptosis Assessment in Rat Brain
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After 72 h reperfusion, the rats were anesthetized and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde. The harvested brain samples (n = 5, each group) were fixed overnight in 4% paraformaldehyde and then sliced into paraffin-embedded coronal sections (4 μm). An apoptosis detection kit (Boster, Wuhan, China) was used to conduct TUNEL staining. Then the samples were coverslipped and examined by an image analysis system (Carl Zeiss, CA, United States).
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