Immunofluorescence

* Frozen Spleen sections * Stain for the marginal zone using IgM (Texas-red) and MOMA-1 )FITC) * Only need 500-1000ul fluid per slide (dependent on section size) 1. 2. 3. 4. 5. 6. 7. Remove slides from -800C freezer allow them to defrost Rehydrate in 1 X PBS 1min (container) Fix-1% paraformaldehyde (made in 1 X PBS) 5min Wash (dip) 3 X in 1 X PBS (container) Block 5% normal goat serum + 1% BSA ( in 1 X PBS) 30 min RT 0C Drain off excess fluid Stain with primary antibodies 1 hr at RT 0C Per slide (make cocktail) 8. 9. 5ul anti-IgM-biotin (1:100) + 5ul anti-MOMA-1 (1:100) in 50ul carrying solution Carrying solution = 1% BSA in 1 X PBS

Wash (dip) 3X in 1 X PBS Stain with secondary reagent (antibody or streptavidin) 45min at RT 0C Per slide (make cocktail) 2ul anti-rat-IgG-FITC (1/1000) + 0.5ul Texas-red-streptavidin (1:4000)

10. 11. 12.

Wash (dip) 3 X in 1 X PBS Drain off excess fluid dont completed dry Mount coverslip use 3 drops of DAKO fluorescent mounting media *apply coverslip from one edge to the other to eliminate air bubbles