Transcription of Protocol 3: Immunofluorescence on Frozen Sections
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Protocol 3: Immunofluorescence on Frozen Sections
Protocol 3: Immunofluorescence on Frozen Sections Brigitte Arduini, version 1, 2015-Mar-23 Modified from Manipulating the Mouse Embryo 3rd edition (2003). Nagy, Gertsenstein, Vintersten and Behringer, ed. Materials Phosphate Buffered Saline (PBS, 1X) Paraformaldehyde (PFA, 4%) see Support Protocol 1 Microscope slides, pre-coated Optimal Cutting Temperature (OCT) Compound 30% sucrose in PBS Cryotome blades (disposable or permanent depending on cryostat) Reagent scoop or slotted spoon Aluminum Foil Conical tube or screw-cap vial Pencil Slide box Humidity box (covered tray with dowels fixed to the bottom) Staining jars/slide holders Cover glass no. TBST solution: 10mM Tris-HCl ( ) + 150mM NaCl + Twe e n 20 50% glycerol solution (diluted in 1X PBS) 4',6-Diamidino-2-Phenylindole ( DA PI, Li f e Te chnol ogi es #D1306) Procedure: Fixation 1. Collect tissue in PBS on ice.
watchmaker forceps if necessary. 5. Add OCT compound if necessa ry to fully cover the tissue sample. 6. Snap freeze the block in methanol/dry ice, using forceps to hold the edge of the foil mold. Frozen blocks may be stored at -80oC for several days, or immediately sectioned. 7.
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