methanol fixation for cell surface marker, the little experiment Re: [Histonet] texture/quality of DAB staining

[Gayle Callis]

I agree whole heartedly with Andrea on using methanol for fixation.

We did a little experiment using
Methanol
Cold acetone 4C 10 min
Acetone 75%/absolute ethanol 25% for 5 min at RT

We did a Phase 2 Coxiella bacteria, immunofluorescent staining using Alexa 
488 as fluorophore.

The staining for both had obvious, dimished intensity after methanol fixation
Acetone for 10 min 4C came in second with diminished intensity, but not as 
bad as the methanol fixation
Acetone/alcohol mixture was decidely the winner with far more cells 
staining and also bacteria.

It is always a good idea to try different fixations.  Caveats about using 
methanol fixation are published by Elias book, have also seen this in other 
literature - that methanol fixation will compromise cell surface staining, 
i.e. CD markers - and this alcohol is probably performing some protein 
hydrolysis which can destroy antigens.   We never use it for endogenous 
peroxidase blocking either, we put our hydrogen peroxide in buffer rather 
than methanol.



   At 03:34 PM 5/5/2006, you wrote:

>(2) Did you try other fixations besides methanol? I find that methanol 
>sometimes can reduce staining for certain
>antigens?

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)