For Tissue Sections
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.
- Paraffin‐embedded tissue slides
- Slide racks & tray
- Staining dishes with lids
- Orbital shaker
- PAP pen & Transfer pipettes
- Deionized water (DI H2O)
- PBS (Phosphate Buffered Saline)
- Primary antibody
- Fluorescent secondary antibody
- Bovine Serum Albumin (BSA – for blocking)
Working Citrate Buffer
- 9mL of 0.1M Citric Acid (10.5g citric acid monohydrate to 500mL DI H2O)
- 41mL of 0.1M Sodium Citrate (14.7g sodium citrate dehydrate to 500mL DI H2O)
- 450mL of DI H2O
Deparaffinization and Rehydration (Cover staining dishes with a lid in each step)
- Dip slides in three (3) changes of xylene or a xylene substitute for 3 minutes each.
- Dip slides in two (2) change of 100% alcohol for 3 minutes each.
- Dip slides in one (1) change of 95% alcohol for 3 minutes.
- Dip slides in one (1) change of 70% alcohol for 3 minutes.
- Rinse slides twice (2x) in DI H2O for 5 minutes.
Antigen Retrieval (Microwave Method)
- Soak the slides in the working citrate buffer and cover with a lid.
- Microwave until the liquid boils, about 1‐5 minutes.
- Remove from heat and let it stand at room temperature for 20 minutes.
- Wash three (3) times for 5 minutes in DI H2O
- Remove the liquid (do not touch the tissue!) and use a PAP pen to circle around the tissue.
- Apply enough 5% BSA with a transfer pipette to cover the tissues.
- Incubate the slides overnight at 4°C in a humid chamber.
- Dilute the primary antibody to the recommended concentration in 1% BSA/PBS diluent.
- Remove the BSA, and incubate with primary antibody solution for 1 hour at room temperature.
- Wash slides three (3) times 5 minutes each on the shaker.
Secondary Antibody and Detection
- Dilute the fluorescent secondary antibody to 1:200 in 1% BSA diluent.
- Incubate with secondary antibody solution for 1 hour at room temperature in dark place.
- Wash slides in PBS three (3) times 5 minutes each on the shaker.
- Apply 50% glycerol/PBS to the middle of the slide.
- Apply coverslip to slide and store slides at 4°C until ready to view.