Cell Array Immunofluorescence Protocol


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

Use separate tubs for antibodies and negative control slides to avoid contamination.

 

MATERIALS

 

REDI-PRO™ cell arrays

Coverslips

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)

Glycerol

 

 

Permeabilize Membrane (Optional)

  1. 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.
  2. Remove the liquid and wash the slides twice (2x) in PBS, 5 minutes each on the shaker.

 

Blocking

  1. Remove the liquid and add 5% BSA into each well. Incubate overnight at 4°C in a humid chamber.

 

Primary Antibody

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

 

Secondary Antibody and Detection

  1. Dilute the biotinylated secondary antibody to 1:200 in a solution of 1% BSA diluent.
  2. Remove fluid and incubate with secondary antibody for one (1) hour at room temperature in dark place.
  3. Wash three (3) times 5 minutes in PBS on an orbital shaker. Remove excess fluid.

 

Cover Slips

  1. Add several drops of coverslip solution (50% glycerol / DI H2O) to the slide.
  2. Place coverslip on top of the slide and store slides at 4°C until ready to view.