4%-PFA Fixed Cell Line Slides
Note: Do not let the tissues dry out once they are re‐hydrated.
Use separate tubs for antibodies and negative control slides to avoid contamination.
- Slide racks & tray
- Staining dishes with lids
- Orbital shaker & Transfer pipettes
- Deionized water (DI H2O)
- PBS (Phosphate Buffered Saline)
- Triton X‐100
- Primary antibody
- Fluorescent secondary antibody
- Bovine Serum Albumin (BSA – for blocking)
Permeabilize Membrane (Optional)
- Add one drop of PBS/0.1% Triton X‐100 to each well to permeabilize the cells. Incubate slides for one (1) minute at room temperature.
- Remove the liquid and wash the slides twice (2x) in PBS, 5 minutes each on the shaker.
- Remove the liquid and add 5% BSA into each well. Incubate overnight at 4°C in a humid chamber.
- Dilute the primary antibody to the recommended concentration in 1% BSA diluent.
- Remove BSA and incubate with primary antibody for one (1) hour at room temperature.
- Remove primary antibody solution and wash slides three (3) times in PBS, 5 minutes each on the shaker.
Secondary Antibody and Detection
- Dilute the biotinylated secondary antibody to 1:200 in a solution of 1% BSA diluent.
- Remove fluid and incubate with secondary antibody for one (1) hour at room temperature in dark place.
- Wash three (3) times 5 minutes in PBS on an orbital shaker. Remove excess fluid.
- Add several drops of coverslip solution (50% glycerol / DI H2O) to the slide.
- Place coverslip on top of the slide and store slides at 4°C until ready to view.