Non-isotopic labeling enzyme detection test - Database & Sql Blog Articles

1. The biotinylated probe is hybridized to the sample on the slide and washed ("Fluorescence in situ hybridization experiment" basic protocol 1~6 steps). 2. Remove the slide from the 1×SSC and blot the buffer on the residual slide as much as possible, but do not dry the slide. Add 200 μl of blocking solution to the slide, place a 24 mm × 60 mm coverslip on the blocking solution, place the slide in a humidified box wrapped with aluminum foil, and incubate at 37 ° C for 30 min. 3. Remove the slide from the humidification box, tilt it to slide the cover slip, and remove the residual blocking solution as much as possible, but do not dry the slide. Add 200 μl of streptavidin solution to the slide and place the cover glass. The tablets were placed on a liquid, and the slides were placed in a humidified box wrapped with aluminum foil and incubated at 37 ° C for 30 min. 4. Remove the slide from the humidification box, tilt to slide the cover slip, place the slide into a Coplin jar containing 42% C. 0.1% Tween 20/PBS, and place the bottle in a shaking water bath at 42 °C. The plate was washed twice with 42 ° C 0.1% Tween 20/PBS for 5 min. 5. Remove the slide and thoroughly remove the residue. Do not dry the slide. Add 200 μl of biotinylated HRPO solution to the slide. Cover it with a 24 mm × 60 mm coverslip and place it in aluminum foil. Incubate at 37 °C for 30 min in a humidified box. 6. Continue with step 4 and repeat the wash. 7. Remove the slide from the wash solution and thoroughly remove the residual solution. Do not allow the slide to dry. Add 0.015% H2O2 to the DAB substrate solution and immediately add 200 μl of DAB substrate solution to the slide. The top cover is covered with a 24 mm × 60 mm coverslip and placed in the dark at room temperature for 10-20 min. 8. The color precipitate was visually discernible and the reaction was stopped by washing with PBS for 5 min at room temperature. 9. If necessary, perform fluorescent counterstaining to identify the nucleus ("Fluorescence in situ hybridization assay", step 9). 10. Cover the slide with 90% glycerol or a suitable anti-fading media and observe or photograph with a phase contrast microscope.