[Histonet] Re: 10% Buffered Formalin Acetate & PBS precipitation artifacts

[Andrea Marion]

Hi all,

This discussion of phosphate buffered formalin has raised some other
questions for me. In paraffin embedding protocols for early mouse embryos,
I have seen recommendations that dehydration begin with steps in 30% and
50% EtOH in 0.9% NaCl or PBS, then the 70% and higher dilutions are in
dH2O.

I was told (and assumed) the purpose was to preserve osmolarity of the
tissue, and minimize cell shrinkage - that this was a 'gentler' way of
dehydrating. However, now it seems to me that at residual PBS
precipitating in the 70% and higher dilutions would damage the tissue?
What about unbuffered NaCl only? What does PBS precipitation artifact look
like?

Has anyone else encountered this practice, or perhaps can shed some light
on the issue?

Andrea Marion
Graduate Student
University of Illinois at Chicago



---
Jennifer, the only reason I am aware of using acetate over phosphate
buffering would be to minimize the precipitation that happens when
phosphate buffered formalin contacts alcohol concentrations greater than
70%. It is why some automated tissue processors have a warm water flush
step. The two fixes to this issue I have heard are:

1 - use 70% alcohol or lower in the step following the last fixative or
2 - use sodium acetate buffered formalin instead

That's my wild guess for the day. I bet if you ask the investigator, the
answer is nowhere close to this. <grin>

Teri Johnson, HT(ASCP)QIHC
Head, Histology and Electron Microscopy
Stowers Institute for Medical Research
Kansas City, MO