[Histonet] Need major help with In Situ . . .

[I.B.]

Hi All,

I am working on a project involving embryonic Sea Lamprey and am having many
difficulties with In Situ.  Namely, the tissue sections (frozen
cryo-sections) are washing off the slides (mostly during the 68oC, 2x and
0.1x SSC washes, but anything that is left washes off before they are
coverslipped), all of them, 100% loss.  Everything worked fine at first, but
then I started losing tissue and have not been able to isolate the problem
and develop a cure.  The embryos were killed, fixed overnight in 4%
Paraformaldehyde, then placed in absolute methanol at -80oC.  Before use I
bring them to water (100%, 95%, 70% ethanol, diH20, 2x 5mins each) then soak
overnight in 20% Sucrose, then soak 4hrs-overnight in OCT compound before
freezing.  After sectioning I dry overnight at 40oC before performing ISH.
I started using Fisher SuperFrost Plus and have tried APES treated slides
(2% APES in acetone for 15min, wash in acetone, wash in diH2O, dried
overnight at 50oC) with no luck.  I have tired working with them laying flat
(the slides, not me) rather than using vertically-situated staining jars,
and using a barrier pen instead of gasketed coverwells to hold solutions
(the suction created while removing the coverwells pulls sections off the
slide).  The latest attempt involved staining/counterstaining the entire
embryo first, then cryo-sectioning and air drying the slides overnight, then
clarifying and mounting.  This worked great, lots of tissue on the slides,
buuuutttt . . .the counterstain (Nuclear Fast Red) washed out while soaking
overnight in 20% Sucrose prior to freezing.  In addition the stain
(NBT/BCIP) looks, well, not good.  Smeared, or spilled (as in, 'spilled out'
of the cells) may be a way to describe it.  So in summary:

Procedure:
In Situ Hybridization
DIG/Alkaline Phosphatase labeled RNA probes
NBT/BCIP stain
Nuclear Fast Red Counterstain
Vectamount Permanent mounting media

Problem:
tissue falling off slides


I am at a complete loss.  Has anyone out there in Histoland worked with
similar tissues or had a similar problem, and how did you fix it?  I have
heard of using RNase A after Hybridization in place of high-temp, high
stringency SSC washes, but have not tired it (we do a lot of work with RNA
in my lab, and I am a bit worried about working with RNase).  Can anyone
recommend this procedure?  Hell, can anyone recommend ANYTHING?  I do not
know how to proceed from here, and your help is dearly appreciated.

IB