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Immunostaining of cells in tissue culture:

    The purpose of fixation is denature the components of cells enough so that they stay on the dish and can be bound by antibodies, hopefully without destroying cellular morphology. Fixatives such as formaldehyde, paraformaldehyde and glutaraldehyde chemically cross-link proteins, by binding to amino acid side chains, generally the most chemically reactive ones like amines (Lysine, Arginine, Glutamine and Asparagine). This chemical modification can also block antibody binding sites, so some antibodies will loose some or all binding activity following these kinds of fixation. Substances such as acetone and methanol are not true fixatives but are denaturants, which precipitate proteins without covalently modifying them. We routinely use a combination of mild formaldehyde fixation followed by cold methanol for neurons, mixed neuron/glial cultures and most of the widely used human and rodent cell lines. The formaldehyde preserves the cellular morphology well, while the cold methanol further denatures the proteins of the cells and helps keep what is left of the cell adherent to the dish. For soluble proteins it may be necessary to miss the methanol step, but in this case you have to be very careful with the washing steps, as the cells tend to wash off the dish. Certain antibodies may be very sensitive to formalin fixation, so you may have to experiment a little, perhaps missing out that step. The following procedure works for antibodies to most cytoskeletal and signaling molecules. This procedure is good for cells in 6 well culture plates or in 35mm dishes. These are just big enough that you can look from above with a typical immunofluorescence microscope through a glass coverslip placed on top of the cells. This allows you to take very high quality high magnification images with lenses up to 100X magnification. However note that it's a pain to changes lenses on the microscope if you use these dishes, since you have to rack the lens up to do this. In the case of the 6 well dishes you also have to remove the lenses neighboring the one you are using since they will hit the other wells of the dish! No procedure is perfect, but note that many of our cell images were made in this way.

1. Draw of culture medium with aspirator and add 1 mL of 3.7% formaldehyde in PBS to the dish. We make this up from 10mLs Fisher 37% formaldehyde plus 90mLs PBS. Fisher formaldehyde is an aqueous solution containing 37% formaldehyde by weight plus about 10% methanol as preservative. Sigma and other companies sell similar material. This formaldehyde/methanol solution is sometimes referred to as "Formalin", a trademark which not all companies are allowed to use. After adding the fixative let the cells sit at room temperature for 1 minute. You can add 0.1% Tween 20 to the PBS used to make the fixative, wash and incubation solutions to reduce background; probably best not to do this first time round though as it may extract your antigen or help wash your cells off the dish.

2. Take off the formaldehyde/PBS and add 1ml of cold methanol (-20C, kept in well sealed bottle in fridge). Let sit for no more than 1 minute.

3. Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add ~10µl (=1%) of goat serum (Sigma), and can incubate for 30 minutes. Can then add antibody reagents. Typically 100µl of hybridoma tissue culture supernatent or 1µl of mouse ascites fluid or crude serum. If you are using a purified antibody a good starting dilution would be 1µg/mL. Incubate for 1 hour at room temp. (or can go at 37C for 30 minutes to 1 hour, or can do 4C overnight, exact time not too critical). Can do very gentle shaking for well adherent cell lines (3T3, HEK293 etc.).

4. Remove primary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS. We usually gently agitate the plates or dishes on a shaker, but you will have to see if this works and/or helps with your particular cells.

5. Add 0.5 mls of secondary antibody. These are fluorescently labeled goat anti mouse or rabbit antibodies and are conjugated to ALEXA dyes. The ALEXA dyes are sulphonated rhodamine compounds and are much more stable to UV than FITC, TRITC, Texas red etc. and are marketed by ThermoFisher. We typically make 1:2,000 dilutions of these from 1mg/mL and add 1% goat serum, BSA or non fat milk carrier to reduce background staining. Incubate for 1 hour at room temp. (or can go at 37C for 30 minutes to 1 hour, or can do 4C overnight). Can do gentle shaking for well adherent cell lines (3T3, HEK293 etc.).

6. Remove secondary antibody and replace with 1 mL of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS.

7. Drop on one drop of Fisher mounting medium onto dish and apply 22mm square coverslip. View in the microscope!


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