Dear Histonetters,

I have a user who is performing in situ hybridization on sections from 
plant floral tissue (Arabidopsis). The problem is that most sections are 
lost during the lengthy procedure. The tissue was embedded in a low-melting 
polyester wax (Steedman's wax) and 5-10 micron sections attached to either 
poly-L-lysine or silane-treated slides.  This has worked great for immunos, 
but in situ hybridization seems too much for those sections to hold on.
The usual tricks of drying the tissue after dewaxing have been applied, but 
without much effect.
So we are contemplating using celloidin (cellulose nitrate) to coat the 
slides with dewaxed sections and thus prevent them from floating away. Do 
you think this is  going to cause excessive background or other problems 
for in situ hybridization?

The protocol I am planning to use is as follows:
1) De-wax the sections on slides as usual (in ethanol, the polyester wax is 
acohol-soluble)
2) Dip the slide in a solution of 50ml  100% ethanol, 49ml ether, 1g 
cellulose nitrate for few minutes
3) Lift the slide and hold in the air few seconds until it is just 
becinning to dry
4) place the slide in 80% ethanol to congeal the nitrocellulose
5) bring the slide to water or buffer through a graded ethanol series


Thanks in advance for your advice
Thank you.
Stan Vitha


Dr. Stanislav Vitha      vitha@mic.tamu.edu
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257

tel: 979-845-1129 (main desk)
tel: 979-845-1607 (direct link)
fax: 979-847-8933




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