[Histonet] RE: Carnoy's fixation and E16 mouse embryos

[gayle callis]

James,

 

You did not say how long you were fixing these somewhat large embryos? Nor
did you give the processing schedule in detail, time/solvent/clearing
agent/paraffin? Please provide this info, thanks! 

 

If the tissues are soft in the center, over dehydration is probably not the
problem. It sounds as though fixation is incomplete, nor are you processing
long enough (under-dehydration plus more) remove water, clear, and
infiltrate with paraffin on these larger embryos OR both of the previous
comments.  If you let your larger embryos sit in this fixative (hopefully a
very large quantity), then the acid may damage the antigens as compared to
using this fixative for a shorter time with smaller, earlier embryos. We
generally used Carnoys, the classic method with 10 ml acetic acid and 60 ml
absolute ethanol and 40 ml chloroform to fix very THIN pieces of tissue
overnight then rinse the fixative out with one change 95% ethanol to get rid
of acetic acid and chloroform.  The processing was started in 95% alcohol
since the fixative also acts like a dehydrant. Acetic acid ruins red blood
cells and could very well damage antigens over a long period of fixation. 

 

If you fixation is incomplete, then the graded alcohols during dehydration
may complete the fixation anyway, not a good thing to have happen.  However,
your tissues are staying soft so inadequate fixation and dehydration may be
factors.     

 

Penetration of the fixative can be improved if you slice skin over abdomen
open to allow fixative into viscera, and also remove tails and appendages
and even head if that is not of interest.   

 

In more recent years, we eliminated the chloroform (too carcinogenic/toxic)
and used only 60 ml absolute ethanol, and 10 ml acetic acid since chloroform
does NOT fix tissue, but is there to remove fats/lipids.  

 

Good luck

 

Gayle M. Callis

HTL(ASCP)HT,MT

Bozeman MT       

 

 

 

 

 You wrote: 

I have been having trouble cutting whole embryo sections on E16 mice. I

think the problem is with the fixation process. I am currently using a

modified Carnoy's fixation (60% ETOH, 30% formaldehyde, 10% Glacial

acetic acid). I would like to use this fixation as I have optimized my

antibodies for this condition. The tissue is soft. I think it is due to

incomplete dehydration. Does anyone have a any suggestions for doing

tissue this large as I know over dehydrating can lead to problems also.