|From:||yichao wu |
Dear Katri,Thank you very much! My answer and question are as below,
>From: " Katri Tuomala" To: "yichao wu"
>Subject: Re: How could I get better frozen slides?
Dear Yichao,Before you embark into any big changes in your protocol, try cutting your sections in warmer temperature. I find that kidney biopsies cut best at -20 C or even warmer. Our protocol of collecting kidney biopsies is very close to yours. How long is the delay from the obtaining the biopsy to freezing it? (3-10minutes)We collect the biopsy, put it onto saline moistened gauze, walk to the cryostat (3-5 minutes), put the OCT onto cryostat chuck (I wonder if the chuck was placed at -20C previously?) and orient the biopsy and let freeze in the cryostat(You mean let them freeze natually by the low temperature in the cyrostat? Do you quick freeze them? What temperature do you use?).
If not cut same day the OCT with the biopsy is detached from the chuck and stored at -80C(Do you put the specimen from -20C to -80C directly? or by some quick freeze method?) until ready to cut and do IF. I don't think you want to use a fixative(Yes,I once want to let the fixative protect the morphology) or you'd have validate your IF staining protocol.
Only problem we have run into occasionally is the too long a delay before biopsy is frozen, resulting in a dried up sections with non specific staining. We do not have freezing artifact as long as you don't immerse the biopsy into saline(Very interesting! Do you mean immerse into liquid should be avoided? just should be transfered in a moistened gauze? ).>My two cents worth, I hope it helps(Surely! Thank you very much!)... Katri > >Katri Tuomala >Department of Pathology >St.Joseph's Health Care >Hamilton, Ontario, Canada