I want to try using DAPI on frozen sections. Is the protocol different from paraffin sections? Should i use triton?
Dear Marwah,
* you should thaw your frozen slides, warm to room temperature, leave for 10 minutes at room temp till sections settles down to surface of slide.
*Wash sections very gently with PBS twice for 5 minutes to get rid of OCT etc.
*Fix sections with some fixative (4%PFA in PBS) for 20 minutes
*Wash sections 2 times with PBS for 5 minutes
*Permealize the sections with "PBS containing either 0.1-0.25% Triton-X-100"
or with PBS with 0.05% Tween-20
*Wash cells in PBS three times for 5 min.
- Add sufficient 300 nM DAPI stain solution to cover the cells.
- Incubate for 5 minutes, protected from light.
- Remove the stain solution.
- Wash the cells 2–3 times in PBS.
- Cover with aqueous mounting media (like glycerol or another available from companies)
- Image the sections, store these slides in dark and 4C
Best of luck
DAPI and/or Hoechst usually work almost all the time. These are common, easy-to-use reagents to stain nuclei. But if you are using frozen sections embedded in OCT compound & sectioned, mounted them onto IHC slide glasses, you just need to wash away OCT compound in axTBS-T for 10 min at RT. Unlike FFPE sections, you need to do xylene & serial EtOH washes at first. Once you remove OCT from the frozen sections, DAPI usually takes 5-10 min to stain at RT. If you do not use Triton-X for your FFPE sections, frozen sections also do not need. Usually, for DAPI staining, Triton-X permeabilization is not required. It can be optional.