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IHC for frozen section for cartilage tissue


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#1 abohollolo

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Posted 06 August 2009 - 02:07 AM

Hi

I am trying IHC for frozen section of cartilage tissue. it was said that it should not fix with PFA or add securose solution in case you want to perform IHC later ... cause PFA mask the antigen (i dont know about the securose)

anyway i tried without PFA and securose and failed the IHC (for collagen type II)

i wounder if the PFA and securose is so critical ? i mean i prefer to use them before cutting ?

any experience please?!

#2 jah

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Posted 06 August 2009 - 03:31 AM

I wonder why frozen section rather than paraffin or resin? The high water content of cartilage causes problems in frozen sections because of ice crystal formation. Sucrose can be helpful in preserving cell& tissue morphology by displacing the water in the tissue. Paraformaldehyde can be helpful or harmful depending on the degree of cross linking and whether the epitope your primary antibody recognizes is affected. For the rare occasion that our lab does frozen preps of cartilage, we first fix 3mm^3 pieces of tissue in 4% PAF (0.1M cacodylate, pH 7.0) for one hour, and then into 30% sucrose/4% PAF in cacodylate overnight. The tissue is then embedded in OCT in tissue molds and frozen in a slurry of Ethanol and Dry ice, and stored at -80'C. We then section at -22'C, adhere to APES coated slide, and then into -20'C MeOH (I keep it in the cryostat while sectioning).

IHC for collagens can also be difficult in any preparation - frozen, paraffin, plastic. You might have to try some antigen retrieval methods, as the tertiary and quaternary structure can be difficult to denature and expose your epitope. Working in order from gentle to harsh, I would suggest starting with citrate buffer(pH 6.5) in a microwave (>90'C for 15min); trypsin/EDTA(15 min) or papain (30min);0.1M HCl x 30 min. Once you get the epitope exposed, you can fine-tune the antibody titer to decrease non-specific background, which increases is more of a problem with the harsher retrieval methods.

Hope that helps!-JAH




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