Tissue Slide Immunofluorescence Protocol


Note:   Do not let the tissues dry out once they are re-hydrated.

Use separate tubs for antibodies and negative control slides.

 

MATERIALS

 

Tissue slides

Coverslips

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)

Glycerol

 

BUFFERS

 

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)

 

  1. Dip slides in three (3) changes of xylene or a xylene substitute for 3 minutes each.
  2. Dip slides in two (2) change of 100% alcohol for 3 minutes each.
  3. Dip slides in one (1) change of 95% alcohol for 3 minutes.
  4. Dip slides in one (1) change of 70% alcohol for 3 minutes.
  5. Rinse slides twice (2x) in DI H2O for 5 minutes.

 

Antigen Retrieval (Microwave Method)

 

  1. Soak the slides in the working citrate buffer and cover with a lid.
  2. Microwave until the liquid boils, about 1-5 minutes.
  3. Remove from heat and let it stand at room temperature for 20 minutes.
  4. Wash three (3) times for 5 minutes in DI H2O
  5. Remove the liquid (do not touch the tissue!) and use a PAP pen to circle around the tissue.

 

Blocking

 

  1. Apply enough 5% BSA with a transfer pipette to cover the tissues.
  2. Incubate the slides overnight at 4°C in a humid chamber.

 

Primary Antibody

 

  1. Dilute the primary antibody to the recommended concentration in 1% BSA/PBS diluent.
  2. Remove the BSA, and incubate with primary antibody solution for 1 hour at room temperature.
  3. Wash slides three (3) times 5 minutes each on the shaker.

 

Secondary Antibody and Detection

 

  1. Dilute the fluorescent secondary antibody to 1:200 in 1% BSA diluent.
  2. Incubate with secondary antibody solution for 1 hour at room temperature in dark place.
  3. Wash slides in PBS three (3) times 5 minutes each on the shaker.

 

Cover Slips

 

  1. Apply 50% glycerol/PBS to the middle of the slide.
  2. Apply coverslip to slide and store slides at 4°C until ready to view.