Tunel HRP—Roche Products
§ Principle: Tunel is a TdT-mediatedX-dUTP nick end labeling method. During apoptotic cells, double-stranded DNA will have many asymmetric breakpoints, exposing 3'-OH, Terminal transferase (TDT) can catalyze the addition of deoxynucleotides labeled with G (homogen), biotin, or fluorescein to the 3'-OH end, and then detect by immunohistochemistry to observe the Morphology, staining location and tissue distribution. Tunel method can be used to detect a variety of samples such as cell suspensions, paraffin / frozen sections, and cell smears.
 
2. Reagent composition: In Situ Cell Death Detection Kit (FITC-HRP), Cat #: 11684817910. 1. Enzyme solution—TDT enzyme (terminal transferase) 5 × 50ul; 2. Labeling solution [FITC-11-dUTP 10 × reaction mixture (contains: 1mM dATP, 1mM dCTP, 1mM dGTP, 0.65mMdTTP, 0.35mM b-Dutp pH6.5)] 5 × 550ul; 3, detection reagent (anti-FITC-HRP existing 3.5ml)
Operation process
Rat adrenal pheochromocytoma cell PC-12 cell climbing test Tunel
1.Cell sheet
   ⑴Cultivate cells adherently, fix with 4% paraformaldehyde for 10-20min, wash with distilled water, and wash with 0.01mol / L pH7.3 PBS for 3 × 3min;
   ⑵ Perforation: wash with 0.1% Triton X-100 / PBS for 5-10min, digest with 20ug / L proteinase K for 5min, room temperature, wash with distilled water for 5minX2, wash with PBS for 3 × 3min;
   ⑶ Preparation of Tunel mixed solution 50ul TDT + 450ul labeling buffer
 ⑷Add 50ul / piece of Tunel mixed solution in a wet box at 37 ℃ for 1h; (this step needs to avoid light)
 ⑸0.01mol / L pH7.3 PBS Wash 3 × 3min;
 ⑹ Add 50ul / tablet anti-FITC-HRP secondary antibody in a wet box at 37 ℃ for 30min;
 ⑺0.01mol / L pH7.3 PBS Wash 3 × 3min;
⑻.0.04% DAB + 0.03% H2O2 for 10min, wash with water
⑼hematoxylin overdose for 1min, washed with blue, series of ethanol dehydration
常规 Sealed with conventional gum after blow drying.
2.Paraffin section
     ⑴ 4 μm paraffin sections are routinely dewaxed to water,
     洗 Wash in PBS 3 × 3min
     ⑶ 3% H2O2 treatment, 20min, room temperature
     Prionase K 20μg / ml 10-20min 37 ℃
     ⑸0.1% Triton X-100 / PBS for 5-10min
     洗 Wash in PBS 3 × 3min
     准备 Tunel preparation 50ul TDT + 450ul labeled buffer
     ⑻30μl / each in a wet box at 37 ℃ for 1h; (this step needs to avoid light)
⑼Wash in PBS 3 × 3min
⑽ Add 50ul / tablet anti-FITC-HRP secondary antibody in a wet box at 37 ℃ for 30min;
   ⑾0.01mol / L pH7.3 PBS Wash 3 × 3min;
⑿ 0.04% DAB + 0.03% H2O2 for 10min, wash with water
⒀hematoxylin overdose for 1min, washed with blue, series of ethanol dehydration
常规 Sealed with conventional gum after blow drying.
4. Observation: Apoptosis index (AI): Observe the number of positive cells counted continuously in 10 high-power fields under ordinary light microscope, and change to the average number of apoptotic cells per square millimeter, which is AI.