Immunofluorescence Protocol for HNF4α (P1 and P2 isoforms)

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Immunofluorescence Protocol for HNF4α (P1 and P2 isoforms)This protocol can be used for immunostaining of mouse liver and intestinal tissues that are formalin fixed, cryopreserved in optimal cutting temperature (OCT) compound and sectioned (at 5um thickness) on to positively charged glass slidesWash the dried frozen tissue sections with phosphate buffered saline (PBS): two times, 5 mins each wash. Don’t let slides dry out after thisAntigen retrieval step for P1/P2 staining: For P1/P2 or P1-HNF4α staining: Immerse slides in 1% sodium dodecyl sulfate (SDS) in potassium-free PBS (pH 7.4) Microwave for 2 minutes at full power, simmer at 10% power for 7 mins (standard 1000 watts microwave oven) or autoclave @121C for 20 minsFor P2-HNF4α staining: Immerse slides in 10mM citrate buffer (pH 6.5). Autoclave @ 121C for 20 mins.Let slides cool down for 15-20 minsWash with PBS: two times, 5 mins each wash Incubate in Na-borohydride (1.5% in PBS) for 15 mins at room temperatureWash with PBS: two times, 5 mins each washIncubate for at least 30 mins at room temperature in GB (Goat Serum: 5%+ Tween-20: 0.05% + Glycine: 0.1% + EDTA: 3mM+ NaCl: 150mM. Adjust pH to 7.4)Wash 5 mins with PBSIncubate in anti-mouse IgG (1:100 dilution) for 4 hrs at room temperature or overnight @4CWash with PBS-Tw (0.1 % Tween-20 in PBS): two times, 5 mins each wash Wash 5 mins with PBSIncubate in primary antibody diluted in GB/PBS overnight @4C (Antibody information: R&D Systems (Minneapolis, MN): P1/P2- HNF4α (#PP-H1415-00), P1-HNF4α (#PP-K9218-00) and P2-HNF4α (#PP-H6939-00). Recommended dilution: 1:300 for P1 or P1/P2 and 1:250 for P2. Dilutions may need to be adjusted for each new aliquot of antibody)Wash with PBS-Tw: three times, 5 mins each washOptional (to stain nuclei): Incubate sections in To-Pro3 (red stain from Life Technologies, Carlsbad, CA; diluted 1:1000 in PBS) for 20 minutes at room temperatureWash with PBS-Tw: two times, 5 mins each washWash 5 mins with PBSIncubate sections in secondary antibody conjugated to Alexa Fluor-488 and/or -546 (diluted 1:1000 in PBS-Tw) for at least 40 mins at room temperatureWash with PBS-Tw: two times, 5 mins each washWash with PBS: two times, 5 mins each washMount in Gelvatol and view fluorescence

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