|From:||yichao wu |
Dear Dr.Calllis, Thank you very much for your kind help all the time.Here is my answer for your question about
>I have a question for you.Are you trying to get good morphology and still do LCM on the same tissue section? If so, you will NOT have the best of both worlds...
Here the biopsy specimens are tiny.If we want to have any glomeruli in every frozen sections,we have to try to combine immunohistochemisty and laser microdissetion together.
I should explain further, the biopies here are aparted for four purposes: formalin-fixed paraffin-embeded sections for light microscopy; acetone/methanol-fixed paraffin-embeded sections for immunohistochemistry (since we found that this kind of fixation maintain antigens well and have very good morphology as well. And we transfered from frozen section to this kind of section to do IHC in 1999); frozen sections and lastly, another part for electron microscope.So the morphology analysis by light microscopy is apart from immunohistochemisty.
Most time it is hard to guarantee there is any glomeruli in the frozen section part.So our director wants that the second part and the frozen section part fusion into one part. That is to say, she wants again to do IHC on frozen sections.But she don't want to give up the good morphology of such paraffin-embeded sections.However she also knows that such paraffin sections are not suitable to do some molecular analysis such as laser microdissection.Hence she is very very hesitate!
So now I am responsible for better frozen sections,especially better morphology.I realized that there may be something wrong with our frozen section protocol.So I asked and get much help from histonet.Now I add "quick-freezing" by liquid nitrogen as you know,and have better results.But it still could not be comparable to paraffin sections.
Therefore,on one hand,we are trying to do laser microdissection on such acetone/methanol-fixed paraffin-embeded sections,wishing to acquire RNA(But we tried and tried and failed). On the other hand, I still want to improve the frozen section protocol further for better morphology to the uppest level.I wish someday my director would like to transfer to frozen sections for both IHC and molecular analysis.Maybe the morphology would be a little worse, but we have better material for laser microdissection and in situ hybridization.
I wish my explanation is clear enough.Sorry for the possible misleading. And I wish you could give me more instructions on my problems.We are really in dilemma!
Thank you very much again!