Re: [Histonet] X-Gal Rat Brain

From:"Andrea T. Hooper"

I work with mouse and human not rat but the principles are the same 
....... For X-Gal staining, PFA isn't the way to go.

PFA will kill your ability to reproducibly and faithfully stain. You 
will likely see less positivity than you should (you might even miss 
positive staining altogether).

For beta-gal/X-Gal staining, you should snap freeze the brains in OCT 
without fixation and fix with 0.2% glutaraldehyde after sectioning 
right before staining (this way you have more flexibility to do other 
things with your other brain sections like immunostain etc as GA 
destroys immunostaining) ... at least for mouse and human and 
morphology is quite good .........

OR you can perfuse with 0.2% glutaraldehyde followed by a 0.2% GA 
submersion fix and then sucrose etc etc.

There is a whole protocol for this as you need EGTA and Mg etc in 
your fixing solutions.

Let me know if you need more info.

>Hi everybody,
>I had a question regarding fixing rat brain. We are currently 
>perfusing and fixing with a 4% para / 3% sucrose solution. I am 
>trying to get X-Gal staining, but I have read this may be 
>undetectable once tissue has been paraffin embedded.  First off, has 
>anybody had luck with paraffinized tissue and x-gal? Secondly, we 
>wanted to snap freeze and section the tissue ourselves if paraffin 
>was not an option.  From what I've tried thus far, I am not getting 
>the structural integrity that comes from paraffinzed tissue.  I know 
>you are never going to get as good slides when frozen, but I think 
>it should look more intact than what I am seeing.  The tissue looks 
>stretched out and very gappy. Could one of the reasons be that the 
>tissue is being overfixed? The tissue has been sitting in the 
>para/sucrose sol'n for anywhere from 3 days to 4 weeks. Maybe my 
>snap freezing technique is off, but I don't think ice 
>crystallization is the problem. Is there any advantage to using the 
>para/sucrose sol'n to fix, or is it just better to fix overnight, 
>move to sucrose gradient and then snap freeze?
>Thanks in advance for suggestions.


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