Hello
Fixation: 4% paraformaldehyde in 0.1M PB or 10% formalin.
The Procedure:
Deparaffinize sections in xylene 2 or 3 changes at 10 minutes each. Hydrate in 100% alcohol 2x5 minutes, 95% alcohol 3 minutes, 70% 3 minutes. Rinse in tap water and then in distilled water. Notes: For 5-10 um sections, 2 changes of xylene, 10 minutes each, will be sufficient. However, for 20-50 um sections, 3 changes of xylene at 10 minutes each, is recommended. Insufficient deparaffinization will cause uneven staining.
Stain in 0.1% cresyl violet solution for 3-10 minutes. Notes: Staining in warmed cresyl violet solution (warm up in 37-50 ºC oven) can improve penetration and enhance even staining. It is particularly beneficial for thicker (20-50 um) sections.
Rinse quickly in distilled water.
Differentiate in 95% ethyl alcohol for 2-30 minutes and check microscopically for best result.
Dehydrate in 100% alcohol 2x5 min.
Clear in xylene 2x5 min.
Mount with permanent mounting medium.
NOT :
# To prepaer 0.1% Cresyl violet solution:
Cresyl echt violet (or cresyl violet acetate) --- 0.1 g
Distilled water ------------------------------------ 100 ml
Add 10 drops (or 0.3 ml) of glacial acetic acid just before use and filter.
# Section: paraffin sections at 5~30 um
References in link
Good luck
http://play.psych.mun.ca/~smilway/fixation.html
http://web.qbi.uq.edu.au/microscopy/cresyl-violet-staining-nissl-staining/
http://www.ihcworld.com/_protocols/special_stains/nissl.htm
Thank you for your exhaustive response. So, 4% paraformaldehyde in 0.1M PB and 10% NFS are the best fixative solution for cresyl violet staining.
In the past I have used Bouin's (picric acid, acetic acid and formalin) this gives very nice contrast between nuclei and cytoplasm. The picric acid presents extra safety issues. The tissue should be rinsed well after fixation. 4% paraformaldehyde in PBS is fine as a general fixative for light microscopy. The other fix that is quite good is Karnovsky's which while developed for EM is very good for light microscopy.
The choice of fix is dependent on how you intend to fix. If by immersion only then Bouin's is fine. It has good penetration. I would not recommend immersion fixation for large brains such as adult mammalian brains; use immersion fixation for fish, frog, embryonic mammalian or excised pieces of adult brain (cortical slices, biopsy etc). If you are using adult mammalian brains, I would recommend perfusion and either paraformaldehyde or Karnovsky's they penetrate well and fix fast.
Thank you all! I`ll use rat embryo`s brain and mouse adult brain. Karnovsky`s... I`ll keep that in mind! Best regards, Vlad.
Yes, I always use 4% fomalin in 0,1 PBS and it gives good result.
the fixation time could be for 24 hrs and also it depend supon the size of the tissue
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