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. 2. Wash 1 2 times in PBS and transfer to a glass vial on ice.
2. Wash 1 – 2 times in PBS and transfer to a glass vial on ice. 3. oCover with 4% PFA and incubate at 4 C for 30 minutes to several hours depending on the size of the tissue. 4. Wash with PBS, 1X fast followed by 2 – 3 times at 4. o. C over the co urse of several hours with slow agitation (such as on a nutator). 5.
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