[Histonet] Frozen Tissue Prep and Fixation Questions for FISH in Spinal Cord and DRG Sections

[patrickbeall <@t> aim.com]

 Hi,
 
 I'm currently working on studying the expression of chemotropic receptor mRNA in neurons during regenration after an injury. I've got a few questions about the best way to prep the tissue with regards to quality and minimizing trauma that may cause confounding results.
 
 1. What is the best way to extract and fix tissue, and is cryoprotection necessary? Initially I followed GeneDetect's frozen tissue protocol (excise tissue from living animal, snap freeze on foil in a -70 Celsius freezer), but I'm concerned about repeated thawing of the tissue producing artifacts (cryosection, thaw mount on slides, air dry, back to the freezer, back to room temp another day, fix in 4% PFA--I imagine I could fix immediately after drying though, if this is otherwise a decent method).
 
 Currently I've been considering just dunking the tissue in a little jar of fix immediately after harvesting, then cryoprotecting in a sucrose based solution prior to cryosectioning. My advisor suggested transcardial fixation, but in that case I'm worried that the anoxia occuring from the time I puncture the diaphragm until the pre-fix wash completes (15-20 minutes) may cause either erroneous RNA expression or RNA degradation. Unfortunately, I don't have much knowledge about the time course of mRNA synthesis and degradation, so it's difficult for me to decide if such a timespan is significant.
 
 2. For spinal or DRG tissue, is it necessary to use anything like proteinase K to make the membrane more permeable when using flourescent anti-sense probes, or is it OK just to hybridize as is.
 
 Any assistance would by greatly appreciated.
 
 Patrick Beall
 NeuroEngineering Laboratory
 School of Behavioral and Brain Sciences
 University of Texas at Dallas
  
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