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

[Eric Eades]

Hi Tim,

It is safe to consider "40% formaldehyde" to be a 100% solution, because
that is the maximum amount of formaldehyde that will dissolve into an
aqueous solution.

Happy Fixation,

-Eric Eades
Phenopath Laboratories
Seattle, WA



On Tue, Jul 24, 2012 at 12:36 PM, Tim Wheelock <twheelock <@t> mclean.harvard.edu
> wrote:

> 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
>
>
>
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