Hi all,

I have a couple of questions:

First, I have a rabbit polyclonal antibody which I believe is 
producing inappropriate staining in my human skin paraffin sections 
due to a cross reaction with epidermal keratins.  Specific staining 
is clearly visible where it should be and in my positive control, but 
I'm getting quite a strong background "blush" uniformly throughout 
the epidermis which I would like to remove.   The dermis is spared.  
Has anyone had any experience in preabsorbing polyclonal primary 
antibodies to remove this cross reaction, specifically preabsorbing 
the antibody with human keratin? Any general tips and pointers 
would be appreciated.

My second problem concerns Methyl Green. I use the VectorLabs 
stain and follow their protocol, heating the stain to 60oC and 
incubating the slides for 1-5 minutes, followed by rinsing in 
deionized water and dipping in acetone/0.05% acetic acid.  The 
staining is rather lighter than I would like but acceptable.  The 
problem comes with the mounting.  I'm routinely using a permanent 
xylene based mount, DePeX from BDH.  This works fine with slides 
stained with haematoxylin.  However, on contact with the sections 
that have been stained with Methyl Green, they completely 
disintegrate!

I know I could try other mountants, but I would like to know why 
this only seems to occur with Methyl Green.

Thanks in advance for any help and also thanks to all those who 
responded to my last question concerning antibody precipitation.  
A combination of spinning the ab for 2 min on max in a benchtop 
microfuge and syringe filtering the chromagen worked a treat.

Liz Sabin.



esabin@srv4.med.ed.ac.uk