Immunofluoresence

Example protocol for staining tissue is below. 

Example protocol for dynamic mammalian cell culture is on next page.

You need to have a sample of the same tissue treated with the same protocol at the same time but with one deletion:  no primary antibody.  You must have this no primary antibody control to ascertain whether positive fluorescence is specific binding or nonspecific background.

Example protocol for immunofluoresence on tissue

I.  IF THE TISSUE IS PARAFFIN EMBEDDED, FIRST REHYDRATE.

1. Xylene --> 100% alcohol --> 100% alcohol --> 95% alcohol --> 70% alcohol --> 50% alcohol  --> 30% alcohol --> water

2. PBS wash

II.  REDUCE BACKGROUND

1. Incubate with NaIO₄ (21.4 mg/ml in PBS) 15 min. followed by PBS wash

2. Alternative bleaching solutions can be fresh NaBH₄ or glycine followed by PBS wash

III.  BLOCK WITH NORMAL SERUM INCUBATION

1. goat serum or powdered milk etc. 20% in PBS 1 hr.

2. blot to remove excess serum

IV.  PRIMARY ANTIBODY INCUBATION

1. in 2% normal serum incubate 4^oC, warm to room temperature, wash with PBS

V.  SECONDARY ANTIBODY INCUBATION

1. Flourescent probe a primary antibody in 2% normal serum/PBS in dark 1 hr.

2. Wash PBS at 4^oC

3. Mount slides in 0.1M n-propyl gallate (Sigma) in gelvatol or in 50% glycerol/PBS and seal edges.

Do controls every time.

If you have any additions, suggestions, or corrections, please let us know.

Electron Microscopy preparation is different and sample fixations can be found here.

last revised 16 May 2001 by mc