Re: Bone and Cartilage Cryosectioning and Staining

From:Elizabeth Chlipala

on 1/16/03 2:29 PM, Lora Huang at wrote:

> Dear HistoNet,
> I usually do Affymetrix GeneChip and spotted array work, but I have an
> investigator with a question that I could not answer, so I thought I would
> try everyone here.  This investigator would like to induce a defect in the
> tibia of rabbits, wait a specific time to allow for healing, and then
> section the bone for use on a Laser Capture Microscope.  The goal would be
> to isolate the RNA of the different cell populations present during the time
> line and then use that RNA on Afftmetrix GeneChips to check for differences
> in expression.
> I tried doing some background research and found a protocol from DeCal
> using their ImmunoCal solution to decalcify the bone and they say that this
> will preserve RNA.  Does anyone know if this will work?
> My other question is how to best stain the bone and cartilage once it
> has been sectioned?  I just need enough stain to be able to differentiate
> between the different cells without killing the RNA.  Any help anyone might
> be able to lend would be GREAYLY appreciated!!  I have only ever sectioned
> organs and tumor tissue, so I'm pretty much helpless.
> Thank you!!!
> Lora Huang
> Univ of Iowa DNA Facility
> ----- Original Message -----
> From: "HistoNet Server" 
> To: "Lora Huang" 
> Sent: Thursday, January 16, 2003 9:59 AM
> Subject: re: subscribe
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We routinely cut sections on decalcified bone to demonstrate cartilage
defects.  We have done this work in the past on mice,  rats, guinea pigs,
rabbits and dogs.  We have used immunocal in the past and formic acid also,
for other clients we have cut RNAse free sections on fomalin fixed and
formic acid decalcified mouse joints.  So I believe that RNA is still
preserved with that process.  You need to limit the fixation and
decalcification process, so if you are using rabbits, the joints should be
sectioned into slabs about 2-3 mm thick upon grossing, fix for 48 hours and
decaled for approxmately  48 to 72 hours (using immunocal).  We use
toluidine blue to stain the cartilage, you will be able to see the defect
and also any other morphological changes in the cartilage with that stain.

Liz Chlipala

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