Patrick, Bring sections out of freezer and air dry at RT immediately, preferably lay on a countertop to warm as quickly as possible. They may look a little "frosty" but let them warm up for a few minutes - what you see is some water condensation. You cannot wash these sections in buffer before fixation, so mark with PAP pen then fix the air dried sections. After that, go to buffer and then stain. IF you wash before fixation, you will lose an unfixed frozen section off the slide.
We prefer to air dry unfixed fresh tissue frozen sections (I presume that is what you did here?) for a minimum of 30 minutes, then put into a storage box that holds ONE days staining run. We put a little bag of 16 mesh silica gel in the box with sections. Take box from freezer, and let the sections come to room temperature for 20 minutes or longer. Open box, mark with PAP pen, fix and stain. If you open the box immediately after removing from freezer, you will get water condensation. And if you open a box, remove some sections, then return box to freezer, you get a thaw/freeze of sections. Water condensation can damage sections and thaw/freeze will eventually damage antigens - I would avoid both. If storing fresh tissue frozen sections as positive and negative controls, use the 5 slide mailers with lids, and take out a mailer to warm up to RT. This keeps other sections frozen until use. We put the mailers w/ slides in a zip lock baggie for quick removal of a few sections to avoid opening and closing a large box. As for BSA, we no longer use it for any immunostaining procedures/buffers/diluents,blocking reagents, etc but if you must use it, there is Jackson's immunoglobulin/protease free. This is also available from Sigma, high quality for molecular biology procedures and in larger quantity. We use only Tween 20 in our buffers now. There is a publication (which I would be happy to send in pdf) about the effects of BSA with primary and secondary antibodies in AIMM journal and why it causes IHC problems. We prefer a normal serum block matched to host of secondary antibody. Once sections are at RT, they stay at RT unless fixed in cold acetone (4C for 10 min, then air dry again at RT) then all staining, etc is done at RT. We never store a frozen section in a cryostat, it goes from pick up to RT for air drying then freezer storage (-80C, -70C is good). Have fun on your new job. Gayle Callis HTL/HT/MT(ASCP) Bozeman MT -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lewis, Patrick Sent: Tuesday, August 24, 2010 3:50 PM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] Quick frozen sectioning and IMHC questions Hi everyone, I cut my first frozen sections, human tonsil, and I have stored them at -70. When I take them out to use, should I bring them to -20C and fix them right away? Or should I bring them to room temp and then pap pen/wash/block/wash them then fix them before adding my primary anti-body. My original plan was/is to bring them to room temp, pap pen them and then wash/block/wash fix. I am asking for your advice as my boss is away for two weeks and the protocol he left me with is a bit vague and requires some modifications to fit the research that we are doing. I was just wondering if there is a compelling reason to keep the sections at/near -20 after sectioning up through the fixation before starting the Immunohistochemistry. Also, we are using BSA as one of the components in our blocking solution as well as a component in our wash solution. Is there a danger in using lower/higher quality BSA? I'm a little worried that our BSA might contribute to increased background if its quality is not high enough. The BSA we may use is A-8327 Sigma, and I am thinking this might be better A3294, or one of these (A9647,A7906,A6793) This is a new job for me, and its been a long while since I have done any Immunohistochemistries.so I am going over the reagents we have on hand while I refresh my memory on immunohistochemistry and sectioning techniques. Thanks for all your support Patrick Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000-0000 PAGER 000-000-0000 CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org <http://seattlechildrens.org/> CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. 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