Hi Jo,
Do dewaxing with xylene and rehydrate with gradient ethanol (100% 2x, 90%, 70%, and distilled water) for 5 min each. Incubate in hematoxylene for 30 sec and wash for 20 min in running tap water. Then incubate in eosin for 30 sec and wash for 20 min in running tap water. Then dehydrate in reverse ethanol gradient and then xylene. Then put cover slide using mounting buffer and keep at RT to make dry. Hope this will help you.
With best.
Hi Jo,
we had good success with the following protocol:
http://sites.igc.gulbenkian.pt/irg/PDFs/HE.pdf
1. Fixation:
- Acetone at -20 °C, for 3 min.
- 80% Methanol at 4°C, for 5 min
NOTE: to mount slides use Permount.
An even more simple staining of cryosections is with 1% Toluidin-blue for 30 sec to 1 min after fixation in 4% buffered formalin.
Regards,
Fred
Fred, thank you! For my clarification, steps 9 and 10 are separate: why? Are the containers separate?
Step 11 is accomplished by air or drip drying? Step 12 is a dip? I am a complete novice and I appreciate all that I am learning from the community!
How soon do you stain cryosections? Mine are in a -20 unstained, in a box.
Thank you, Fred,
Jo
Hi Jo,
To confirm that the slides are completely dehydrated you need to use xylene 2 times that I mentioned in my answer as 2x. It should be in two separate xylene chamber and ensure that these chambers are not the same you used in rehydration process.
I agree with Fred but different lab has different established protocol. In my opinion, you don't need to follow step 11 and 12 as you already incubated slides in xylene for 2 times (5 min each). Be also careful that the slides shouldn't be dried after xylene incubation. Put mounting buffer as soon as possible when take out the slide from xylene and put cover slip.
With best
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