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Protocols - Immunofluorescence: Rabbit Anti-Murine Leptin

The following protocol used Trichuris muris infected murine tissue. The tissue was fixed in 4% PFA, then embedded in paraffin, and cut into 5 μM sections.  

  1. Deparaffinize and rehydrate the tissue section.
  2. Perform heat-induced antigen retrieval by boiling the tissue section in 10mM pH 6.0 citrate buffer for 25 minutes.  
  3. Incubate the tissue section with blocking buffer for 20 minutes.   
  4. Incubate the tissue section overnight at 4˚C with Rabbit Anti-Murine Leptin at 0.2 μg/mL in 1X PBS with 0.01% Triton-X and 0.5% BSA.  Wash the slide twice for three minutes (1X PBS/0.05% Tween 20).
  5. Incubate the tissue section with a fluorescent conjugated secondary antibody for 2 hours at room temperature.  Wash the slide twice for three minutes.
  6. Counterstain the tissue section with DAPI. 

*Information courtesy of David Artis, University of Pennsylvania.