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From:"Shelton, John" <shelton@ryburn.swmed.edu>
To:Histonet <histonet@pathology.swmed.edu>
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Ms Suarez,

I have successfully done in situ for two of the genes you are intersted in, however not in human.  I have done the mouse homolg of FLT-1 and FLK-1 with good results in mouse placenta and embryonic mouse heart, respectively.

The FLK-1 work is part of a manuscript just submitted describing a Cre mutation of several genes involved in angiogenesis; since the work is still in review, I cannot provide a reference for you.

The FLT-1 work is another story, and can be found in EMBO 1999(18): 5943-5952.  Only scarce details of the in-situ are included in the methods of this paper, my apologies.

My standard protocol worked well, using hybridization O/N at 55C and 50% formamide.  Lengthy high-stringency washes with 50% formamide at 65C and lengthy low-salt washes at 37C produced "good" staining.

Some suggestions:
 Paraformaldehyde instead of formalin
 Duration of fixation kept to a minimum
 Human tissue,  then use probes from human database
 Start with a good positive control rather than archived blocks
 Take steps to assess the quality of your probe
 Take steps to assess the RNA integrity and availability in your blocks
     If you cant get replacement tissue for making blocks of known quality,
     this lengthy effort will tell you if the RNA is available in your archive
     1.) Perform your deparaffinization and prehybridization washes as usual
     2.) Set up an RT-PCR reaction on the section using a hyb-aid adhesive
             chamber spacer, plastic coverslip, and RTh
     3.) Aspirate the reaction solution from the chamber and analyze for correct
             sized DNA.

I know you have received alot of suggestions.  If you still find yourself with no success or not knowing which steps to take to correct the problem, call me. Pull the EMBO reference and see what is obtainable with the mouse homolog.

John Shelton
Research Scientist
UT Southwestern

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