ICC Main Page > Antibody Staining
The third step of cell staining involves incubation of cell preparations with antibody. Unbound antibody is removed by washing, and the bound antibody is detected either directly (if the primary antibody is labeled) or, indirectly using a enzyme-labeled or fluorochrome-labeled secondary reagent.
1.1 Add 1 ml of the washed cell suspension to a 15 ml conical centrifuge tube and pellet the cells by centrifuging for 5 min at 200g.
1.2 Decant the supernatant, and add 50-100 ul of primary antibody diluted in PBS (Note: antibody concentration should be determined by titration of the stock solution and testing on a known positive specimen. Usually, working concentrations are in the range of 10-20 ug/ml. However, depending on the source this concentration could vary significantly).
1.3 Gently re-suspend the cells in this small volume by flicking the bottom of the tube with a finger.
1.4 Incubate on ice for 30 minutes.
1.5 Wash the cells three times in PBS, centrifuge for 5 min at 200g, and decanting the supernatant.
1.6 Add 50-100 ul of fluorescence (FITC, Taxes Red) conjugated secondary antibody and incubate on ice for 30 minutes.
1.7 Wash the cells three times in PBS, centrifuge for 5 min at 200g, and decanting the supernatant. After the last centrifugation, re-suspend the cells in two drops of PBS (Note: it is important to keep the cells concentrated at this stage to facilitate examination of an adequate number in the next step).
1.8 Mount the cells on a slide with a coverslip, and examine on the fluorescent microscope with the appropriate filters for fluorescein excitation and emission (Note: antibody molecules will not penetrate the cell membrane of living cells, so any cell showing fluorescence throughout its interior has died. If the cells are not kept cold or maintained with 0.1% sodium azide, capping will occur; antibody-antigen complexes will clustered into one region of the cell membrane resulting in a crescent-shaped fluorescent labeling pattern).
2.1 Blocking: Wash cultured cells attached to tissue culture dishes in PBS, then incubate in a blocking buffer consisting of BSA-PBS for 5 min, and cool to 4 C. Cooling prevents subsequent endocytosis of any added antibody reagents, as well as minimizing lateral mobility of bound antibody in the plane of the plasma membrane.
2.2 Primary Antibody: Add the primary antibody to the living cells at 4 C in PBS, and incubate for 30 min. Do not pipet directly onto the cells, but add antibody solutions at the edge of the dish, and add wash solutions from a wide-mouth bottle or beaker to minimize the potential of removing cells by too vigorous a fluid steam. Do not allow the cells to dry at any step. Rock the dish back and forth to maintain coverage over the cells if the antibody solution volume is too small (Note: the most sensitive approach to surface antigen labeling is to incubate cells at 4 C while alive with antibodies. This prevents internal labeling background and also minimizes artifacts seen with some fixation. However, there are situations in which living cells are not available or the antigen of interest fails to react without some form of fixation. In these settings, formaldehyde fixation or other forms of fixation can be performed prior to incubation with antibodies).
2.3 Washing: Pipet out the primary antibody solution from the dish. Wash the dish again 3x5 min with PBS at 4 C.
2.4 Secondary Antibody: Add a fluorescent conjugated secondary antibody in PBS and incubate the cells at 4 C for 30-60 minutes.
2.5 Pipet out the secondary antibody and wash the dish 3x5 min in PBS at 4 C, followed by washing briefly in PBS at room temperature.
2.6 Fixation: Fix the cells attached to the dish in 3.7% formaldehyde-PBS at room temperature for 15 min (Note: the final formaldehyde fixation step links all of the antibody steps in place, preventing their dissociation. Immediate viewing of cells may not require this step, but after fixation, mounted cells can be kept at 4 C for several weeks with little loss of signal. Drying at any stage will severely affect the label, either causing it to b3e displaced or drastically altering morphology of the surface).
2.7 Mounting and Coverslipping: Wash the dish 3x5 min in PBS at room temperature, cover the cells with anti-fade mounting medium, and overlay with a coverslip.
3.1 Fixation: Cultured cells attached to tissue-culture dishes are washed in PBS, then fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. The dishes are then washed 3x5 min in PBS (Note: coverslips or glass slides can also be placed in the bottom of individual tissue-culture dishes for cell growth when a slide format is desired. Formaldehyde works well as a primary fixative in this setting because it preserves cell morphology well and the time of exposure to the fixative is short. Formaldehyde fixation at room temperature is very effective, but at 4 C, it is very poor fixative. Another fixative approach is the use of organic solvents, such as ethanol, methanol, and acetone. These precipitating fixatives also produce membrane permeability and generally yield a poorer quality of preservation, although with a high degree of permeability).
3.2 Blocking and Permeabilizing: Incubate dishes with blocking solution containing 0.5% Triton X-100 for 30 min at room temperature.
3.3 Primary Antibody: The primary antibody diluted with antibody dilution buffer or PBS is then added to the fixed cells and incubated for 1-2 hours at room temperature (Note: do not pipet directly on to the cells, but add antibody solutions at the edge of the dish, and add wash solutions gently at the edge of the dish to minimize cell detachment.
3.4 Washing: Decant the primary antibody solution, and immediately wash the dish 3x5 min with PBS. Do not let the cells dry at any step. Especially during washing, handle each dish individually, since leaving a washed dish without medium for even a few seconds can allow drying in the center of the dish.
3.5 Secondary Antibody: Add the fluorescent (FITC, Texas Red) conjugated secondary antibody in PBS and incubate for 30-60 minutes at room temperature.
3.6 Decant the secondary antibody and wash the cells 3x5 min in PBS.
3.7 Post-Fix: Fix the cells again using 3.7% formaldehyde freshly made as performed in the initial fixation. The purpose of the second fixation is to crosslink the antibodies in place and to prevent subsequent diffusion of label. If not postfixed in this way, the localization may not be stable for more than a few hours.
3.8 Wash the cells 3x5 min in PBS.
3.9 Mounting and Coverslipping: Mount the cells with anti-fading medium with coverslip.