H&E staining - method (Mar/01/2007 )
i'm new to histology. I need to do some staining on fresh tissues. Have bought Eosin Hematoxylin solution from sigma. But i could find any protocol for it.I searched in web, found two different methods. Pls help me to choose them. Pls suggest if have your own protocol.
Method 1: Deparaffinize and rehydrate sections. (First xylene, followed by ethanol)
Method 2: Rehydrate first, use H&E stain, later go for xylene.
More over all the protocol has Eosin and Hematooxylin separetly, not togethar.
In my experience, you always need to deparafinize in xylenes first. But if you want to mount with a non-aqueous mounting medium such as permount, then you need to go back to xylenes after the H&E staining. Here's a link to a protocol similar to the one I use, but I only do 3 minutes for each change of the xylenes and EtOH.
For your specific H&E, I would call sigma tech support to make sure of the staining times and exact protocol.
Each and every H&E protocol is not the same but can be similar. Check with sigma for their recommendations.
We deparafinise in xylene, down the ethanol and into water, then into hematoxylin and then into eosin and up the ethanol ladder and into xylene before mounting the slides.
hi i checked with sigma,here is the protocol .but I have new doubts.
1. What is the use of lithium carbonate
2. Is there any alternative to that.
1. Place in stain from the 95% alcohol. 1-2 minutes is usually sufficient.
2. Remove each slide, blot and rinse in up 95% alcohol until the excess stain has been
3. Transfer slides to bluing solution (lithium carbonate probably best) and leave until blue.
4. Rinse 15-20 min. with running water.
5. Counterstain if desired. Dehydrate, clear and mount.
histoclear for twenty minutes makes for a less toxic deparafiniseation (is that even a word?).
i dont know about a eosin haemotoxylin soln but we used to ;
1) de parafin (aka - take to water)
4)eosin (everything else - ie it is the counterstain to haemotoxylin)