EZQuant–ELN Knowledge Base

Immunocytochemistry	Table of Contents

Fixation:

• Grow cells on glass coverslips or chamber slides.
• Gently pull off media and wash once with sterile ice-cold PBS.
• Pull off PBS and add 4% formaldehyde in PBS for 45 min at room temperature
  (Use chemical hood).
• Wash with PBS.

Cell Permeabilization (for staining of intracellular antigens):

• Pull off PBS.
• Immerse in PBS-Tween (PBS-T; PBS containing 0.2% Tween 20) for 15 min at room temperature.
• Wash 5 min with PBS on a rocker (250rpm).
• Wash 10 min with PBS+2% BSA on a rocker (250rpm).

Blocking:

• Pull off washing solution.
• Incubate with blocking solution for 30 min on a rocker (250rpm) at room temperature.

Primary antibody:

• Pull off blocking solution.
• Incubate with primary antibody for 1 hr on a rocker (250rpm) at room temperature.
• Wash 3 x 10 min with PBS+2% BSA on a rocker (250rpm).

Secondary antibody:

• Pull off washing solution.
• Incubate with biotin/fluorescence-conjugated secondary antibody for 1 hr on a rocker (250rpm) at room temperature.
• Wash 3 x 10 min with PBS on a rocker (250rpm).

Avidin (if necessary):

• Pull off washing solution.
• Incubate with HRP/fluorescence-conjugated avidin for 1 hr on a rocker (250rpm) at room temperature.
• Wash 5 x 10 min with PBS on a rocker (250rpm).

DAB (if necessary):

•   Pull off washing solution.
•  Add DAB in peroxide buffer - Monitor color development.
•    Wash 2 x 5 min in ddH₂O.
•   Dehydrate:
  1 min ethanol 70%
  1 min ethanol 95%
  1 min ethanol 100%
  2 x 5 min Xylene (in a chemical hood).
• Place cover slip, with cell layer down, on a glass slide containing a drop of mounting medium.
• For fluorescent stains, skip the dehydration and use anti-fade mounting medium.