ImmunoCytoChemistry
Solutions and Reagents
1. 1X Phosphate Buffered Saline (PBS)
2. 10% Normal Goat Serum (NGS) in PBS
3. Triton-X (0.2%, PBS) – To prepare 5 ml, add 100 µl 10% Triton to 4.9 mL PBS)
4. NH4Cl (100mM, PBS) – To prepare 50 ml, add 50 mL PBS to 0.267g NH4Cl
5. DAPI (1 ug/mL, PBS) – To prepare 1 mL, add 1 uL of 1 mg/mL DAPI to 1 ml PBS
6. Mounting medium
First day protocol
1. Cell are already fixed ( washed in PBS, 20 min in 4% PFA, washed and left in PBS)
2. Aspirate PBS and add 200 ul Triton-X (0.2% PBS), 5 min
3. Aspirate, wash with PBS
4. Aspirate, add NH4Cl (100mM, PBS), 10 min
5. Aspirate, wash with PBS
6. Aspirate, incubate in 10% NGS (PBS) for 30 min
7. Aspirate, add primary antibody solution (10% NGS, PBS + antibody) – O/N
Second day protocol
7. Cells in primary antibody solution
8. Aspirate, wash with PBS x3
9. 200 ul of secondary antibody solution (PBS + antibody) – 1h
10. Wash in 200 ul DAPI (1ug/mL, PBS) – 10 minutes
11. Remove DAPI solution with pipette and transfer to a “DAPI waste” marked falcon
tube (discard in the end to DAPI waste glass bottle in acid cabinet)
12. Wash with PBS 2x (no aspiration, transfer to “DAPi waste” falcon)
13. Mount slides
 Mark the glass slides (cell line, experiment #, antibodies (name, color)
and date)
 Put a drop of mounting medium on 1 or 2 places on the glass slide
(approximately 10 ul, but load more into the pipette to avoid bubbles)
 Remove the cells-containing coverslip with the fine tweezers, making
sure to not scrape too much off it
 Wash in dH2O prepared in a falcon tube, carefully not to detach the
cells
 Touch the tip of the coverslip on a Kimtech whipe paper and grab the
edges with fingers (letting go the tweezers) and roll the cover slip 360
degrees
 Grab with tweezers again and put on mounting medium on glass slides,
face down (avoid making bubbles)
 Put the slides in the drawer (dark) at room temp. O/N to dry
 Transfer tomorrow to the fridge