We use a 3:1 ration of cold acetone/100% ETOH to fix frozens for IHC. Slides are always air dried first to remove any moisture. Usually 75 mls acetone,25 mls alcohol.
Bernice Frederick HTL (ASCP) Senior Research Tech Pathology Core Facility Robert. H. Lurie Cancer Center Northwestern University 710 N Fairbanks Court Olson 8-421 Chicago,IL 60611 312-503-3723 b-freder...@northwestern.edu -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Gayle Callis Sent: Thursday, March 12, 2015 12:02 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Acetone fixing and tissue damage You wrote: Hi Everyone. When I fix my cryosections in acetone, I am using HPLC grade 99.9% for 10 minutes at -20C. Would the Histology grade 99.5% be less damaging to them? Higher H20 content, i.e. less than 99.5% apparently is also very bad. With the HPLC grade I often get tissue damage, the tissue also floats off the slide causing a stringy effect. Fixing with 4% p-formaldehyde or 100% Methanol, prevented the antibody from recognizing the Nuclear Antigens. Looking for advice, Patrick. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 **************************************************************************** ********************* HPLC grade acetone is not necessary plus very expensive. Use ACS Certified Reagent 99.5% grade, not histology grade, which you can buy in gallon size. Maybe what you are calling histology grade is the ACS Certified Reagent grade but "Histology" grade implies a practical grade of acetone which is not a pure as the ACS certified Reagent grade. Also, 4°C acetone works just as well. If you are storing your acetone (in a staining jar) inside the cryostat to maintain a 20°C temperature, don't!!! If your staining container tips over, you will ruin your cryostat!! Hopefully you are using high quality plus charge slides? We had frozen sections come off a plus charge slide after single 4°C acetone fixation on occasion. You can prevent frozen section loss is a Double Acetone fixation that also increases permeabilization. An IHC guru gave me this hint years ago and was given to her by a company selling immunostaining products. A small fan will be your best friend for RT air drying and/or evaporating acetone. However we air dried all FS were dried at RT for 30 minutes minimum or longer before fixation. By air drying, you get rid of the water. You can put your FS in front of a small fan, or inside a hood for faster drying, and never store just cut FS in the cryostat where water condensation occurs when you take them out of cryostat environment to RT. DRY frozen sections were the rule in our lab before any solvent fixation. 1. Air dry frozen section at RT for 30 min 2. Fix FS in 4°C acetone for 10 minutes 3. Air dry FS for 15 minutes to evaporate acetone 4. Return FS to 4°C acetone for 10 minutes 5. Air dry to evaporate acetone 6. Proceed to immunostaining Gayle Callis HTL/HT/MT(ASCP) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet