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Immunofluorescence Protocol for HNF4α (P1 and P2 isoforms)

KC Karthikeyani Chellappa
PD Poonamjot Deol
FS Frances M Sladek

Last updated date: Jan 12, 2024 Views: 1420 Forks: 0

original An abbreviated version of this protocol was published in eLife in May, 2016
Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer
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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 slides

  1. Wash the dried frozen tissue sections with phosphate buffered saline (PBS): two times, 5 mins each wash. Don’t let slides dry out after this
  2. Antigen retrieval step for P1/P2 staining: 
    1. 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 mins
    2. For P2-HNF4α staining: Immerse slides in 10mM citrate buffer (pH 6.5). Autoclave @ 121C for 20 mins.
    3. Let slides cool down for 15-20 mins
  3. Wash with PBS: two times, 5 mins each wash 
  4. Incubate in Na-borohydride (1.5% in PBS) for 15 mins at room temperature
  5. Wash with PBS: two times, 5 mins each wash
  6. Incubate 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)
  7. Wash 5 mins with PBS
  8. Incubate in anti-mouse IgG (1:100 dilution) for 4 hrs at room temperature or overnight @4C
  9. Wash with PBS-Tw (0.1 % Tween-20 in PBS): two times, 5 mins each wash 
  10. Wash 5 mins with PBS
  11. Incubate 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)
  12. Wash with PBS-Tw: three times, 5 mins each wash
  13. Optional (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 temperature
  14. Wash with PBS-Tw: two times, 5 mins each wash
  15. Wash 5 mins with PBS
  16. Incubate 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 temperature
  17. Wash with PBS-Tw: two times, 5 mins each wash
  18. Wash with PBS: two times, 5 mins each wash
  19. Mount in Gelvatol and view fluorescence    
Copyright: © 2024 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Chellappa, K, Deol, P and Sladek, F(2024). Immunofluorescence Protocol for HNF4α (P1 and P2 isoforms). Bio-protocol Preprint. bio-protocol.org/prep2551.
  2. Chellappa, K., Deol, P., Evans, J. R., Vuong, L. M., Chen, G., Briançon, N., Bolotin, E., Lytle, C., Nair, M. G. and Sladek, F. M.(2016). Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer. eLife. DOI: 10.7554/eLife.10903

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