laser microdissection on frozen sections and paraffin-embeded sections

From:yichao wu

Thank you all the experts who have helped me so much!And especially Gayle Callis and Tony Henwood.
Now I use liquid nitrogen to quick-freeze specimens before cryosectioning and I really get much better morphology.My purpose is to microdissect specimens by Leica laser microdissection system.Now I found frozen sectioned tissue are not easy to stick to the membrane-based slides offered by Leica.Such kind of membrane-based slides are their new product which have metal borders.I think maybe the membrane is too thin to hold the tissue by "temperature difference".How do you use this kind of slides to make frozen sections?

Another question is,since the frozen sections are not as good as paraffin-embeded ones in morphology.I wonder who have successfully acquired RNA for RT-PCR from laser microdissected tissue on paraffin sections?I have tried and tried and failed.The kits I used are "Qiagen Rneasy Mini kit"(I have no "Micro kit" at hand) and one from arctrus and one from Gentra called puroscript.And after Dnase digestion,it seems that I have not acquired any RNA from paraffin embeded sections.The fixation is acetone-methanol(1:1).I wonder if such fixation would destroy RNA? And I wonder if heating sections after paraffin embeding is destroyable? How are other procedures should be followed for microdissection on paraffin embed sections? Pls help me out  Thank you very much!

And by the way,do you think disposable blazes are better than steel-based knife when sectioning specimens from renal biopsies?

With my best regards,

Yichao WU, Ph.D candidate 

Jinling Hospital,Nanjing University,Nanjing, P.R.China

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