[Histonet] Bielschowsky questions, Luxol Fast Blue answers, and Protocol Drift.

[Tim Wheelock]

Hi Everyone:

I had a few questions regarding Bielschowsky silver stains.

(1) What adhesive (if any) or type of slide do you use for the stain?
(2)  How do you clean the glassware?
(3)  When diluting the 40% formaldehyde when making up the developer, do 
you consider the 40% formaldehyde as 100% and then dilute it down by 
using 1 part formaldehyde and 9 parts distilled water?
       Or do you assume the 40% formaldehyde is 40% and then dilute it 
down using 1 part formaldehyde and 3 parts distilled water?
       (My protocol may have inadvertently changed from the first method 
to the second; I am not sure.)
     

By the way, I want to thank everyone for helping me solve the problem 
of  my Luxol Fast Blue staining the myelin too lightly.
I discovered that somehow, I had started adding twice the amount of 
acetic acid to the Luxol staining solution as I should of.
(This protocol "drift", where a mistake can actually find its way into a 
written protocol, can  be a real problem in a lab, especially  when 
working for  years by oneself, as  I have) .

But I also found that even reducing the acetic acid, while helping a 
lot, did not completely fix the problem.
I needed to switch from staining the tissue for 2 hours at 60C  to a 
full over-night (why I never needed to switch times before is a 
mystery). That  did the trick beautifully.
The myelin is staining perfectly again.

Thanks again,

Tim Wheelock
Harvard Brain Bank
Belmont, MA