Microscopy (Part-III)

Lab Experiment 33.1: Determine the Phagocytosis of beads in J774A.1 macrophage cells.

Material and equipments

1. Methanol
2. Acetone
3. PBS (1X)
4. 1% Triton X-100
5. BSA (Fat free, acetylated): Prepare 5% BSA solution in PBS and filter with the 0.45mm filter to rmove particulate matter.
6. Primary antibody (anti-protein): An antibody can be developed against protein (antigen of interest) in rabbit or mice.
7. Secondary antibody: An antibody coupled with fluorescent marker (such as FITC) and directed against mouse IgG.
8. mounting medium.
9. 1µm Latex Beads
10. Filipin: Prepare 5mg/ml stock solution of filipin in 100% alchol. The working solution is 50µg/ml in PBS. 
11. Glass slides
12. Cover Glasses: 12mm circular cover glasses available from the vendor are polished and are not suitable for cell attachment. Cover glasses are washed with alchol and allow the cover glasses to air dry. Keep the cover glasses in a 50ml glass beaker and wrap with the alluminium foil. Autoclave the cover glasses to avoid contamination during phagocytosis experiment.
13. Forcep: Autoclave the forcep to avoid contamination during phagocytosis experiment.   
14. Epi-fluorescence microscope