[Histonet] anti-bromodeoxyuridine staining

[Kitchell, Marianne]

I am having a problem with my anti-bromodeoxyuridine (BRDU) stain. I
have done this for many years on chicken tissues fixed in Notox. 

Now I have rat tissues (Notox again) and sometimes I get everything
staining (all nuclei) and sometimes I get next to nothing to stain. I
have recently started cutting all my sections (paraffin) at 4 um.
Instead of 5um which may be a partial effect. 

I am pretty sure that my problem is in the 2.0 N Hydrochloric acid (HCl)
step and/or the trypsin step. 

It seems that if I use freshly prepared HCl, I get a better response
than if I have previously prepared and stored my 2.0 N HCl or buy 2.0 N
HCl prediluted. This in turn affects the amount of time required in the
subsequent trypsin step. Every time I get a new bottle of HCl I need to
retitrate the whole stain. Any help here so I am not redoing this into
infinity? I am starting to live the movie Groundhog Day.

I have never been able to perform this stain without an additional
trypsin step even though I have seen it done this way in the literature.

Thank you all in advance.

 

 

Marianne Kitchell

Novus International

St. Charles, Mo.

 


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