Hoechst staining method 1. Adherent cell Acid-Base Balance of Medicine,Acid-Base Balance,Calcium Gluconate Injection,Finished Pharmaceutical Product,Acid-Base Balance Agent NOUVASANT GROUP LTD. , https://www.nouvasant.com
1) Take ordinary clean coverslips in 70% ethanol for 5 minutes or longer, dry them in a sterile clean bench or wash them three times with cell culture PBS or 0.9% NaCl, then wash them with cell culture solution. Again. The coverslips were placed in a six-well plate and seeded into cell culture overnight to approximately 50%-80% full.
2) After stimulating the cells to undergo apoptosis, the culture solution is aspirated, and 0.5 ml of the fixing solution is added and fixed for 10 minutes or longer (over 4 ° C overnight).
3) Remove the fixative and wash twice with PBS or 0.9% NaCl for 3 minutes each time to drain the liquid. Shake it with a shaker or shake it several times by hand.
4) Add 0.5 ml of Hoechst 33258 staining solution and stain for 5 minutes. It is also advisable to use a shaker or shake it several times manually.
5) Wash twice with PBS or 0.9% NaCl for 3 minutes each time.
6) Drop a drop of anti-fluorescence quenching on the slide and cover the cell cover with a cell cover to avoid air bubbles. Keep the cells in contact with the sealing solution and do not reverse it.
7) Fluorescence microscopy can detect blue nuclei.
2. Suspended cells
1) Centrifuge the collected cell samples in a 1.5 ml centrifuge tube, add 0.5 ml of fixative, and slowly suspend the cells for 10 minutes or longer (over 4 ° C overnight).
2) Centrifuge the fixative solution and wash twice with PBS or 0.9% NaCl for 3 minutes each time. Manually shake during washing.
3) After the last centrifugation, remove most of the liquid and retain about 50 ml of liquid, then suspend the cells slowly, and add them to the slides to make the cells evenly distributed.
4) Dry it slightly so that the cells stick to the slide and do not easily flow with the liquid.
5) Evenly drop 0.5 ml of Hoechst 33258 staining solution and stain for 5 minutes. Use a blotting paper to remove the liquid from the edges and dry it slightly.
6) Wash twice with PBS or 0.9% NaCl for 3 minutes each time.
7) Apply a drop of anti-fluorescence quenching to the slide and cover with a clean cover slip to avoid air bubbles.
8) H. Fluorescence microscopy can detect blue nuclei.
3. Tissue sectioning
1) After routinely burying the slices, dewax and transparent.
2) Wash twice with PBS or 0.9% NaCl for 3 minutes each time, draining the liquid. Shake it with a shaker or shake it several times by hand. It can be operated in a six-well plate.
3) Add 0.5 ml of Hoechst 33258 staining solution and stain for 5 minutes. It is also advisable to use a shaker or shake it manually.
4) Wash twice with PBS or 0.9% NaCl for 3 minutes each time.
5) Place the slice on the slide, drop a drop of anti-quenching sealant, and cover with a clean cover slip to avoid air bubbles.
6) Fluorescence microscopy can detect blue nuclei.