Protocol for immunofluorescence staining of adhesion cells

This is provided as a general protocol. Optimization of concentration or incubation condition of

the primary antibody and the secondary antibody for your own specimen is necessary. Please

also review the datasheet of the antibody and publications using the same antibody for reference.

Preparation

Coverslip preparation:

 # 1.5 coverslip. In general you should use #1.5 coverslips as most microscope objectives

are designed to work optimally with these.

 Size of coverslip. Round 18 mm coverslips in a 12 well plate work well, or 12 mm

coverslips in a 24-well plate, or 22 mm square coverslips/25mm round coverslips in a 6-

well plate/35mm culture dishes.

 Sterile coverslip. Washing in ethanol or exposing to UV for 60 min.

 Coating of coverslip. Many cell lines will grow well on uncoated coverslip but some do

better with coated coverslips (e.g. 50 µg/ml poly-lysine for 1 hr, or collagen).

Cell preparation:

 Transfer your cells on the coverslip in plates/culture dishes and culture them (e.g.

overnight) so they are well adhered and reach 50-70% confluency.

Reagents preparation:

Note: Always filtration or centrifuge solutions before use to avoid precipitation.

 Phosphate-buffered saline (PBS)

 BSA or Serum from the species that the secondary antibody was raised in

 Fixatives: Formaldehyde/Paraformaldehyde; Methonal; or Acetone

 Note: Choose the appropriate fixation method according to the application.

 The goal of fixation is to maintain cellular structure but may damage or mask

antigenic sites. You may need to test several fixatives before you find the one that

produces the right balance of antibody binding and structural integrity of the

sample.

 Formaldehyde/paraformaldehyde and glutaraldehyde are aldehyde fixatives which

cross link proteins and generally preserve cell structure well but cause strong

autofluorecence, especially glutaraldehyde. This can be counteracted with

reduction of the aldehyde groups with NaBH4 or NH3Cl at 10 mM (See the step

of Quenching). 4% formaldehyde/ paraformaldehyde for 10 min is a good starting

point for mammalian cells. 4% paraformaldehyde + 0.1% glutaraldehyde is good

to preserve cell structure but quenching is required before mounting.

 Organic solvents like methanol precipitate proteins, which produces a similar

outcome to crosslinking with aldehydes but all the small molecules within will be

lost during the rest of the protocol. Fixation with methanol denatures proteins, so

never use if you have a fluorescent protein that you want to look at in your sample.

Using a combination of methanol and acetone can improve immunolabelling lost

in 100% methanol fixation (methanol is best for structure, acetone permeabilizes

well and is less damaging). Try 10 min in solvent at -20°C as a starting point.

 Triton/NP-40/Digitonin or Saponin

 Permeabilization helps the antibodies get into the fixed cells. Cell surface proteins

don't require much/any permeabilization. If the target protein is expressed

intracellularly, it is very important to permeabilize the cells.

 Triton X-100 is the most popular detergent for improving the penetration of the

antibody. However, it is not appropriate for the use of membrane-associated

antigens since it destroys membranes.

 Prepare primary-antibody in 3% BSA/PBS, dilute antibodies according to the

recommended manufacturer specification data sheet or reference publications.

 Selection of the secondary antibody is depended on the donor species of the primary

antibody and the desired fluorochrome.

Procedure

Note: Incubate samples in the dark and cover whenever possible.

It is advisable to run the appropriate negative controls. Negative controls establish background

fluorescence and non-specific staining of the primary and secondary antibodies.

 Rinse cells with PBS x2

 Fixation: fix the cells either in cold methanol, acetone (1-10 min) at -20

C or in 2-4%

paraformaldehyde (PFA) (10-20 min) in PBS (freshly prepared) at RT.

 Wash the samples with PBS 10min x3 on shaker

 Permeabilization: incubate the samples for 10-15 min with 0.05-0.25% Triton X-100 in

PBS (or 100 μM digitonin or 0.5% saponin).

 Wash the samples with PBS 10min x3 on shaker

 Blocking: incubate the cells for 30-60min with 1-3% BSA in PBS to block unspecific

binding of the antibodies (alternative blocking solutions are 1% gelatin or 10% serum

from the species that the secondary antibody was raised in).

 Remove the blocking buffer by holding each coverslip on its edge with forceps and

draining it onto a sheet of Kimwipes.

 Primary Ab: Incubate cells the primary antibody dilution in 3%BSA/PBS (keep cells

dark in a humidified chamber) 1-4 hr at room temperature/37

C or overnight at 4

 A simple chamber can be made from a small box with water-soaked filter papers.

 If it is desirable to examine the co-distribution of two different antigens in the

same cell, a double immunofluorescence procedure may be used. Cells may be

incubated simultaneously with two primary antibodies, provided they are

monospecific and can be distinguished with secondary antibodies conjugated to

different fluorochromes (or with primary antibodies directly conjugated to

different fluorochromes).

 Wash the samples with PBS 10min x3 on shaker

 Secondary antibody: incubate cells with the secondary antibody in 3%BSA/PBS for 30-

60min at RT (keep cells dark in a humidified chamber). Most secondary antibodies can

be used after 1:200-1:500 dilution

Note: If the primary antibodies are already conjugated to a fluorochrome, incubation with

secondary antibody is not necessary.

 Wash the samples with PBS 10min x3 on shaker.

 Counter staining: stain nuclear with DAPI or Hoechst for 5-10min.

 Wash the samples with PBS 10min x3 on shaker.

 Quenching: To quench auto-fluorescence, incubate coverslips with 1 mg/ml Sodium

borohydride (NaBH4) in PBS for 5min. (This solution is prepared immediately before

use.) The solution will foam when PBS is added, so use a large enough tube to avoid

overflow.

 Wash the samples with PBS 10min x3 on shaker

 Mounting: mount coverslip up site down on slide with a drop of mounting medium

 Remove the blocking buffer by holding each coverslip on its edge with forceps

and draining it onto a sheet of fiber-free paper

 Remove any excess liquid from the coverlip and apply 1 drop (30-50ul)

(depending on the surface area of your sample) of mounting medium on the slide.

 Carefully lower the coverslip onto the mounting medium to avoid trapping any air

bubbles.

 Allow the mounted sample to cure on a flat surface in the dark. Curing time may

vary from a couple of hours to overnight, depending on the thickness of the

sample and the relative humidity of the surrounding air.

 For long-term storage, seal the coverslip to the slide after curing to prevent

excessive shrinkage of the mounting medium, which can result in sample

distortion. After sealing, store the slide upright in a covered slide box at 4°C.

 To view the samples immediately, secure the coverslip using nail polish or hot

wax to prevent the coverslip from moving.