[Histonet] Fetal Tissue - In Situ - Fixation Issues

[Emily Sours]

Having done both chick and mouse embryonic nervous tissue, I would suggest
letting it fix for 2 - 4 hours (2 hrs if it's under E11.5).  This may help.
If it doesn't, try fixing the embryo longer--perhaps it takes longer for the
fix to perfuse as the embryo gets older--remember, there are more tissues
for the fix to absorb through.
Also, you may want to lower your concentration of proteinase K in your in
situ--using it at room temperature for three minutes is always a good digest
for our chick embryonic tissue using PK from Roche (embryos fixed from  2 to
overnight) , but we have left it as long at five minutes.  Note that we do
not heat it to 37 degrees Celcius but leave it at room temperature, and make
it right before this step.  This also brings up the fact that you may be
using your proteinase K repeatedly--which is not bad, unless you freeze/thaw
it.  That is not good for proteinase K--for every aliquot, you should freeze
it, then thaw it once (throwing it away afterwards) for optimal results.
The next possibility I would consider is that someone is being too rough
with your samples.  It may sound ridiculous, but I can't tell you how many
times that has come into play when processing our slides (can I emphasize
GENTLE, DON'T DROP!).  Every step requires gentle lifting from the solution,
and gentle lowering into the next. Furthermore, use enough solution to cover
the slides COMPLETELY AND MORE; use enough solution to at least cover 1 cm
of distance between the top of the slide and the meniscus of the liquid
level.  I haven't calculated some magical number between the two; it seems
that a separation affects the way tissue adheres to the slide, as if near a
surface tension--my guess only.


-- 
Yog-Sothoth knows the gate.
Yog-Sothoth is the gate.
Yog-Sothoth is the key and guardian of the gate. Past, present, future, all
are one in Yog-Sothoth.
He knows where the Old Ones broke through of old, and where They shall break
through again.