[Histonet] RE: IHC cartilage - long response

[Elizabeth Chlipala]

Victoria

Cartilage can be tricky, proper processing and sectioning is important initially.   The samples can sometimes be cartilage only or both cartilage and the underlying bone, we have worked with both. It also depends upon what species you are working with.  We have worked with mouse up to horse bone/cartilage samples and have run a lot of IHC on these types of tissue samples.  You need to initially make sure that the tissue is fixed properly and then if there is underlying bone then the decalcification process needs to complete.  We will primarily use a formic acid based decal but on occasion we will use EDTA - this is antibody dependent .  For bone and cartilage samples I like to fix for at least 48 hours if you are working with a larger animal species such as sheep, goat, canine or horse its ideal if the sample can be cut into slabs around 3 -4 mm thick, don't cut them too thin or else you will run into the tissue popping out of the block when you try to section them.   We have worked with larger tissue sizes such as the medial/lateral femur and medial/lateral tibia so larger sample sizes are possible too.  Longer processing cycles are required and then when sectioning you need to use a good plus slide and if processed properly these samples should section just like any soft tissue sample.  We cut at 5 microns and dry flat on a hot plate.  For the IHC (depending upon the antibody) we will initially pilot enzyme retrieval such as proteinase K, pepsin, trypsin but we may also try hyaluronidase or chondroitinase too.  If none of those work then we will try HIER but at 70C for 2 hours rather than a higher temp for a shorter period of time.   It can be tricky but will work well in most cases.  Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz <@t> premierlab.com
www.premierlab.com

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Victoria Rimkunas
Sent: Monday, February 03, 2014 11:29 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC cartilage

Does anyone have experience or a protocol for IHC staining on cartilage?

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