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| A shared resource available for researchers with appointments in the Vanderbilt University Medical Center, supported by the Vanderbilt-Ingram Cancer Center, the Diabetes Research and Training Center, the Digestive Disease Research Center, the Kennedy Center, the Vision Research Center, and the Mouse Metabolic Phenotyping Center. |
Basic Protocol for Fluorescence Immunohistolabeling of Tissue SectionsTissue pieces dehydrated in ascending ethanols, embedded in paraffin.Serial sections made on conventional microtome, stuck to "charge+" slides. De-paraffinize in xylene; 2 x 15 min (all done in Copelin jars at RT). Wash in 100% EtOH; 2 x 5 min. Re-hydrate in ascending ethanols: 95,70,50,35% x 5 min ea. (can sit at 70% indef.). Rinse in H20; 2 x 5 min. Rinse in PBS; 2 x 5 min. Apply Pap- Pen to briefly dried circles around samples (creates hydrophobic wells). Rinse in PBS. Permeablize in PBS / 0.1% Triton >= 10min. Rinse in PBS. From here, use volumes to cover wells made by Pap - Pen (200-500 ul). Incubations are done in humid chambers to minimize evaporation. Block with PBS / 5% N.S. / 1% BSA (use normal serum of your 2' ab, eg donkey) >= 20 min. Pour off; add 1' Ab O/N. Typically 1 ug/ml, (depends on Abs) in PBS 0.1% Triton / 1% BSA. Wash 2 x 10 min in permeablizing medium. 1 x 10 min in PBS. Done in Copelin jar. Incubate with small volume of 2' Abs (mixed) for 1 hr, around 0.5 ug/ml. Wash 2 x 5 min in perm. med. 1 x 5 min in PBS. For Hoechst, try 1:500 dil'n of 1 mg/ml (in PBS) stock for 20 min, check brightness. Wash 1 x 5 min in perm. med. 1 x 5 min in dH20. Cover sample with a drop of AquaPolyMount, tap out bubbles, press flat. Dries in minutes to semi- solid gel, ready to image. Doesn't hurt to seal edges with nail polish or wax. Abs: 1' Abs should be of different species, 2' Abs should be the same. 2' stocks are made up to 1 mg/ml in PBS, then diluted 1:1 in glycerol, and kept in freezer. Can be used 1:1000 (1:250 for Cy-2). 1' stocks are more variable, kept in fridge in various dilutions, use at 1:1000 final dilution. Example: 1': rabbit anti- glucagon is at 1:5, so dilute 1:200 from stock in fridge ~.4 ml / sample. 1': guinea pig anti- insulin is at 1:10, so dilute 1:100 from stock in fridge. 2': Cy-2 donkey anti- rabbit is in box 13 in freezer, dilute 1:250 because it's less bright & photostable. 2': Cy-3 donkey anti-guinea pig is in box 8 in freezer, dilute 1:1000, and mix 2' Abs. 9/5/97 JHS
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