[Histonet] Decal following ISH?

[Gayle Callis]

Dear Nancy, 

Try using EDTA after ISH.   Small embryos with shells will probably take
very little time in 14% Tetra Sodium EDTA in PBS (we use Dulbeccos PBS)
adjust the pH down to 7.4 - 7.6 with glacial acetic acid although some use
hydrochloric acid.  We adjusted the pH using constant stirring on a magnetic
stirrer with single electrode immersed in solution allowing continuous
readout on the pH meter.   Basically, you are titrating the pH down with
acid using a plastic Pasteur pipette, very easy.    Tetra Sodium EDTA is
very alkaline, hence adjusting pH down with one of these acids is necessary.
7.4 is normal pH for Dulbeccos PBS and 7.6 is a working pH for TRIS buffered
Saline (TBS) or a use a pH compatible with ISH protocol.    

EDTA will not be bothered by samples immersed in alcohol and you may be able
to preserve the ISH work done.  It is worth a try.   In the future, I
suggest the researcher do fixation, then EDTA decalcification before ISH
particularly if the signal is ruined on snails with shells.  I bet it would
make his results better too - no shell to deter penetration of reagents.
Good luck and let us know if you have success.   

Happy Holidays!!!   

Gayle Callis
HTL/HT/MT(ASCP) 

-----Original Message-----
From: Thomas, Nancy via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, December 20, 2016 6:10 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Decal following ISH?

Hello all,
I received whole mount samples in 70% ethanol for paraffin processing.  The
samples are snail embryos and the researcher already did in-situ on them.
Because they are of varying ages, some of the shells will section without
decal, but some will need it.  However, this step was not done before the
ISH staining.  Does anyone know if decalcification can follow an ISH
procedure?  I have searched a few protocols online, and each of them did the
decal before hybridization.  I have not yet found one where the decal
followed the procedure.   My plan is to start with one embryo and try it to
see what happens.  Maybe the signal will remain, or maybe not.  Has anyone
done it this way and know the answer before I try?
Thank you,

Nancy Thomas
Senior Lab Manager, Histology Core
Stowers Institute for Medical Research
1000 E. 50th Street
Kansas City, MO  64110

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