Immunofluorescent detection

Immunofluorescent detection of BrdU, Insulin, and other antibodies

Slide Prep

• Pre-mark where the islet sections are located on the slide so they ca be found later
• Heat slides at 60C for 15 min (hybridization oven)
• De-paraffinize and re-hydrate:
• Xylene 2x5 min
• 100%EtOH 2x2 min
• 95%, 90%, 80%, 70%, EtOH each for 1 min
• ddH2O 2x2 min

 Antigen Retrieval & Blocking

• Make antigen unmasking solution by adding 7.5 ml of solution to 800ml of ddH2O. Place slides upright in 1000ml glass beaker, microwave 13.5 min.
• Run under cool tap water for 30 min to cool
• PBS wash 3x5 min
• Using PAP pen, encircle the islet or pancreas sections
• Block for 30 min at room temperature with serum-free blocking reagent (ours is 5x, so dilute it appropriately)
• PBS wash, 3x5 min

Primary Antibody Incubation

• For each section on the on a slide, prepare 80ul of the antibody mixture per islet section or 150-200ul per pancreas section (use serum-free blocking reagent)
• Mix and add the solution to the section. Incubate overnight at 4C in a humidified chamber

Secondary Antibody incubation 

• PBS wash, 3x5 min - For each section on a slide, prepare 80-200ul of secondary antibody solution (make a minimum of 500ul)
• Add 75-150ul of secondary antibody solution per section for 1 hr, room temp, humidified, and dark.
• At this point, keep slides away from light at all times

Post-fix, nuclear counter stain/mount

• PBS wash, 2x5 min
• Add DAPI nuclear counter stain (1:1000 in PBS) for 5 min
• PBS wash, 2x5 min
• Add 2 drops of FluorSave Reagent and apply coverslip. Store in dark until ready for viewing with fluorescent scope