[Histonet] RE: Nissl on Thick Paraffin...
|From:||"Due, Brice" (by way of histonet)|
Hello Nissl, I deleted your original post by accident & histosearch.com appears
Try John Kiernan's modified cv nissl. It works more progressively than the
cv nissls I know, which are purely regressive. 100ml 0.1% cresyl violet spiked
with the addition of 0.5ml of 1% oxalic acid. See p124 of his book.
I do nissls on 15-20um paraffin, not the 40um you're talking. Even with John's
oxalic nissl, I still like to overstain and regress with rosin gum. Gives a
cleaner background = higher contrast. The rosin is a messy sticky sticky
procedure, but you can control the differentiation by diluting the rosin.
After aqueous cv staining of your choice, dehydrate slides in 95% etoh. Use
1-10% rosin gum in 95% etoh to differentiate. Make a 10% stock and cut when you
need finer / slower control. You need a couple changes of rosin soln because it
gets dirty fast. Dip slides in rosin until stain starts running, then rinse in
95% etoh and check on an old scope. Repeat until you get the contrast you want.
If you take out too much cv, just re-hydrate and start over.
The main problem with ultra-thick sections is that the front exposed side
tissue will differentiate faster that the back side which is "hidden" against
the slide. I would try diluting your differentiator (whatever procedure you're
using) 1:10 and see if you can gain some control that way. Are free-floating
sections an option? I've never tried that with paraffin embedded stuff. Or
try a non-cv based progressive nissl. I don't have a good recommendation there.
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