[Histonet] Thionin Staining Recipe and Protocol

[Amanda Madden]

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