[Histonet] ORO staining in pre- vs. post-fixed tissue

[Rene J Buesa]

Michele:Methodologically it is much more difficult to freeze/section formalin fixed tissue so it is unnecessary adding an additional layer of difficulty to the procedure pre-fixing in formalin.Although I have not tried it, there is no effect of NBF on the amount of fat so there should not be a difference in the reaction of ORO.I think that you have presented this question probably because you have old already fixed tissue samples and now want to use ORO on them and if this is the case, they will be very difficult to make frozen sections on them.The best ORO procedure is to "embed" the unfixed tissue in OCT → make FS → fix on NBF vapors → stain with Harris hematoxylin → stain with OROPlease not that in my procedure nuclear staining precedes ORO stain to assure better nuclear detail, and there is no interference at all with the fat stain.Fixation should be done with NBF vapors, not liquid. To do that I always placed the water washed and dried FS (that usually are near one end of the slide) inside a Coplin jar with a small amount of NBF at the bottom in a way that the section does not touch the NBF. The capped Coplin jar is placed in an oven at 60ºC during 15 minutes, so the section is fixed by the vapors. Then the section is washed and the staining is performed.René   

    On Wednesday, January 27, 2016 5:28 PM, Michele Wich via Histonet <histonet at lists.utsouthwestern.edu> wrote:
 

 Does anyone know whether there is a difference, quantitatively, in Oil red O fat staining of tissue fixed in NBF prior to embedding in OCT vs. fresh frozen tissue embedded in OCT? I know that sections from fresh frozen tissues must be post-fixed in formalin before staining with Oil Red O. But is there any benefit to using this preparation over tissues that are fixed in formalin prior to OCT embedding (aside from sectioning/slide adhesion issues previously described)? Is the staining similar, or does one preparation give better staining results?
Much gratitude for any advice you can give,
Michele

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