[Histonet] CD10, CD103, FDA, fatty tissues, complement,
frozen blocks, fixation
JPCOLEMA <@t> sentara.com
Wed May 12 13:02:43 CDT 2004
For CD10 we use Novo Castra's antibody at a 1:30 dilution, Trilogy from Cellmarque (high pH, edta, tween) for 15 minutes in a 98C water bath, and biogenex's super sensitive concentrate kit for detection. 45 minute primary antibody incubation.
Question- I need a reliable CD103 antibody for formalin fixed paraffin embedded material. The clone we have currently is good on coagulative fixed and air dried material, but is worthless on routinely proccessed bone marrow and lymphoid tissue.
Pardon my cynical response, but FDA doesn't care if it works. If you follow the protocol you're in. If you tweak the protcol you're not in FDA land and can not claim such, even if your controls look great.
For fatty tissues we have a fixative that is 4 gal formalin,1 gal absolute ETOH and 500 ml Glacial acetic acid . We place the blocks in this after the tissue is grossed in at the board.
Complement- we stain for C3c, C4c, and C1q on FFPE kidney and skin biopsies using .1% protease from sigma for 30 minutes @ 40C before DIF labelling.
Does your cryostat have a defrost cycle? if so the blocks are being trashed. I recommend sealing in oct, wrapping in parafilm and storing in a -70 freezer or processing as usual and doing immunos on formalin fixed tissue, where the morphology is better.
My 2 cents on fixation- tissue is fixed when it's fixed.(Formalin penetrates and fixes 2mm per hour , right?) It's not fixed because the pathologist wants the tissue out early. I think the sixteen hours is overkill myself and IHC retrieval methods are in place to counteract long fixation. If it were my wife I would cut the tissue in at 4 mm, fix for 8 hours and run the tests like I do with all my patients. I've optimized my lab to give every single person the best possible result. My wife or daughter or mother is no more or less valuable than anyone else that walks through the door. If anyone would do differently for people important to them in their lab then you guys need to bring your standards up! This is medicine- someone's life litereally depends on our results. If your routine methods are not good enough for your family you should be ashamed to leave them in place.
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