Simple CV or Thionine method:
Cresyl Violet or Thionine0.1 gram
Distilled water100 ml
when dissolved add
10% acetic acid0.5 ml
*or *
glacial acetic acid100 µl
1. Bring paraffin sections to water as above, frozen or cryostat
sections are rinsed in water.
2. Stain 2-10 minutes in 0.1% cresyl violet or thionine.
3. Rinse in 2 changes of distilled water, 30 seconds to one minute each.
4. Dehydrate quickly in a few dips in 70% ethanol then 95% ethanol, 15
seconds, then 2 changes of 100% ethanol, one minute each.
5. Clear in 2 changes of xylene and coverslip with DPX mounting medium.
Some stain will come out in the lower alcohols which is normal. Only
nuclei and Nissl should stain. Sounds like your stain is too concentrated.
OR, if you prefer a buffered solution ...
1. Bring paraffin sections to water.
2. Stain 20 minutes in:
0.1% aqueous *Cresyl Violet* or *Thionin*, one part.
0.2 M Walpole acetate buffer, pH 4.05, ten parts.
(16 ml 0.2M acetic acid + 4 ml 0.2M sodium acetate)
3. Rinse twice in distilled water, 30 seconds to one minute each.
4. Dehydrate quickly in 95% ethanol, 15-30 seconds, then 2 changes of
100% ethanol, one minute each.
5. Clear and coverslip.
Source:_Staining Procedures_, 4th edition., George Clark editor.
Geoff
On 3/28/2012 10:56 AM, Amanda Madden wrote:
Good Morning Histonetters!
I am looking for advice on my thionin staining recipe and protocol. The
recipe I use currently ends up being just under 1% Thionin. It is made by
mixing 80ml 1M acetic acid and 14.4ml 1M NAOH to a pH of 4, and then adding
305.6ml 1.3% stock thionin. When I used this protocol last, I rehydrated
the mounted rat brain sections (perfused with 4% buffered para) through
ETOH to dH20 steps, then left the slides in thionin for 6 minutes, followed
by 2x2min rinses in dH20, 1 min in 70% ETOH, 3 min in 95% ETOH, and finally
absolute ETOH and xylene for coverslipping. The tissue came out too dark,
and what I am wondering is if I should change the thionin recipe to
something a bit more dilute, or if I should focus on the baths following
thionin, for instance only rinsing in water and moving into a .1% acetic
acid in 70% ETOH differentiator? Any help, either by directing my own
troubleshooting or even recipes and protocols would be greatly appreciated!
Thanks, as always, for being such wonderful resources for those like myself
who have such little histology background :)
Amanda Madden
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583
mcaul...@umdnj.edu
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