Frozen Tissue Sections
1. Snap freeze fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embed in OCT compound in cryomolds. Store frozen blocks at – 80C.
2. Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at – 80C until needed. The slides can be stored at -20C for short term storage (a few weeks).
3. Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air-dry for 30 minutes.
4. Wash in distilled water and proceed to standard staining procedure.
Whole Animal Paraffin Sections
1. Deeply anesthetize mouse or rat with pentobarbital sodium (Pentane).
2. Fix kidney by transcardial perfusion, first with 50 ml of phosphate buffered saline (0.02M PBS pH 7.4) containing heparin (5U/ml), then with 220 ml of ice-cold 4% paraformaldehyde on 0.1M PBS pH 7.5 containing 4% sucrose.
3. Embed kidney block in paraffin, cut at 7 um intervals and mount sections on slides.
Single Organ Paraffin Sections (For best result perfuse whole animal)
1. Place dissected organs in labeled 20-ml snap-cap glass vials. Fill vials with freshly prepared 4% PFA fixative at 4°C.
This general protocol is is intended for use as a reference as a courtesy to our customers. Optimal concentrations, experimental conditions and experimental processes are to be determined by the individual user. No guarantee of performance using the above procedure is expressed or implied. Molecular Innovations does not currently perform immunohistochemistry in-house and can not provide further technical support for this application.
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