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.
*Information and photo are courtesy of David Artis, University of Pennsylvania.
- Deparaffinize and rehydrate the tissue section.
- Perform heat-induced antigen retrieval by boiling the tissue section in 10mM pH 6.0 citrate buffer for 25 minutes.
- Incubate the tissue section with blocking buffer for 20 minutes.
- 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).
- Incubate the tissue section with a fluorescent conjugated secondary antibody for 2 hours at room temperature. Wash the slide twice for three minutes.
- Counterstain the tissue section with DAPI.