Tip: Keep all mechanical manipulations to a minimum to avoid compromising the quality of the final cell images. Always treat the cells and coverslips gently, never let them dry out and avoid dropping solutions directly on the cells.
Seed the cells on sterile glass coverslips. Some cell types may require growth on poly-lysine or other treated coverslips for proper adhesion.
Tip: Coverslips can be sterilized by dipping in ethanol and flaming them or by placing them into tissue culture dishes and exposing to UV radiation (present in most tissue culture hoods). Several small circular coverslips can be seeded in a single dish to reduce the number of dishes in your incubator.
Grow the cells to semi-confluency.
Aspirate the culture medium from the dish or remove each coverslip as required with tweezers, and gently wash with PBS at room temperature.
Incubate the coverslips in freshly prepared 4% paraformaldehyde - PBS at room temperature for 10 minutes. Alternatively, the cells can be fixed in -20°C methanol for 10 minutes.
Tip: Alternative fixations should be tested and compared to determine which is best at preserving the structure and epitope of the protein of interest within the cell.
Wash the coverslips of fixative in PBS for 2 minutes.
Incubate the coverslips in 0.5% Triton X-100 in PBS at room temperature for 5 minutes. The final working percentage of different detergents (ex. digitonin, Tween-20) should be explored to find the optimal conditions to best preserve the cell structure and protein of interest.
Tip:Permeabilization step is not required when fixing cells using methanol.
Wash the coverslips of the permeablization buffer by incubating in PBS for 5 minutes.
To reduce the background fluorescence, block the coverslips in 1-5% normal serum or BSA prepared in PBS for 1 hour at room temperature.
Tip:The normal serum block should be of the same species in which the secondary antibody has been raised.
Prepare primary antibody dilutions for ICC in 1% normal serum or BSA. Typical working concentrations of antibodies may range from 5-20 µg/mL, however each antibody may require optimization to obtain an appropriate signal.
Tip: Directly conjugated antibodies eliminate the need for secondary antibodies (and hence reduce background) and shorten the time required to complete the ICC procedure.
Incubate the coverslips with the primary antibody dilution for 1 hour at room temperature (37°C is optional), or overnight at 4°C.
Tip: Make sure the coverslips do not dry out during the antibody incubations.
Wash the coverslips gently in PBS three times for 5 minutes each. Additional or longer washes maybe required if excessive background remains.
Prepare an appropriate dilution of fluorochrome-conjugated secondary antibody in 1% normal serum or BSA. Typical starting dilutions range from 1-2 µg/µL, however optimization may be required for the best image.
Incubate the coverslips in the secondary antibody dilution for 1 hour at room temperature in a dark environment.
Wash the coverslips gently in PBS for three times 5 minutes each. Additional or longer washes may be required if excessive background remains.
Tip: Double labeling experiments (2 different antibodies from 2 different species) are best carried out by sequentially incubating with the primary and secondary antibodies. Make sure the primary antibodies are from differing host species, and if this option is not available, directly conjugated primary antibodies may be employed. The use of fluorescently labeled phallodin is a good alternative to immunostaining actin as a reference marker in ICC.
MOUNTING AND IMAGING
When all the necessary washing steps have been completed, the coverslips can be counter stained with DAPI or Hoechst (1-10 µg/mL) to stain the nuclei.
Invert the coverslip onto a glass slip with a drop of mounting media containing a fluorescence antifade agent.
Carefully remove the excess mounting media if necessary and seal as required with nail polish.
Tip: Some mounting media solutions have DAPI already added and will harden after exposure to air, eliminating the need to seal the edges of the coverslip.
Examine the cells under a fluorescence microscope and image as required.
Tip: Avoid long exposure to the excitation wavelength of the fluorochrome to prevent photobleaching.
If too much background fluorescence is present in your samples try the following suggestions:
Increase the percentage of blocking agents used in the blocking step and in the primary and secondary antibody dilution buffers. Increase the time of blocking and/or reduce the time of antibody incubations.
Titrate the amount of primary and secondary antibody used in all the incubations.
Some cells may contain high amounts of your protein of interest in the cytoplasm (ex. tubulin and actin). If antibodies to these proteins are used in your ICC procedure, it can result inexcesive staining. The level of cytoplasmic staining can be reduced by a brief extraction of the cytoplasm in 0.1% Triton X-100 prior to fixation.
Try quenching the fixation agent to eliminate any free aldehyde groups which could non-specifically bind antibody. Quench formaldehyde with 0.1M Tris or Glycine buffer and quench glutaraldehyde with 0.1% sodium borohydride.
Dropped a Coverslip
If a coverslip of cells is accidently dropped and “cell side up” orientation is lost, you can still salvage the experiment by carefully picking up the coverslip with tweezers and gently scraping one surface with a pipette tip to see if any cells are visibly removed.