ICC-IF Standard Protocol
Standard protocol for immunocytochemistry with fluorescent detection optimized for Triple A Polyclonals™ and PrecisA Monoclonals™ from Atlas Antibodies.
Download a pdf version of the standard protocol for ICC-IF.
In addition to the primary antibodies, a set of reference markers are used in each image displayed in our product catalog: an antibody-based marker against microtubules as well as the nuclear dye DAPI.
Perform all washes at room temperature.
- Coat a multiwell, glass-bottomed plate (Greiner bio-one, product no: 655891) with fibronectin (concentration 12.5 μg/ml) for 1 h at room temperature.
- Seed the cells (10,000-25,000 cells per well) and incubate at 37°C in humified air with 5.0% CO2, for 20-24 h.
- Remove the growth medium and wash the cells in Dulbecco’s Phosphate Buffered Saline (DPBS).
- Fix for 15 min in 4% formaldehyde (methanol-free) in DPBS.
- Permeabilize for 10 min with 0.1% Triton X-100 in DPBS.
- Wash with DPBS once and incubate for 1 h at room temperature in blocking buffer (4% FBS in DPBS) to block non-specific sites.
- Incubate the cells overnight at 4°C with primary antibodies in blocking buffer.
- The following day, remove the primary antibodies and rinse the cells briefly in DPBS. Then wash with DPBS 3x10 min.
- Incubate for 1.5 h at room temperature with secondary antibodies in blocking buffer.
Note: The secondary antibodies are fluorescently labeled and thus light sensitive. Keep the sample protected from light.
- Counterstain for 5 min with the nuclear stain DAPI (Invitrogen) at 1ug/ml in DPBS.
- Wash with DPBS 3x10 min and mount in DPBS containing 0,02 % (w/v) NaN3.
- Triple A Polyclonals at a working concentration of 0.25-2 µg/ml.
- PrecisA Monoclonals at a working concentration of 2-10 µg/ml.
- Mouse anti-alpha Tubulin monoclonal antibody Ab7291 (Abcam) diluted 500x for Triple A Polyclonals.
- Rabbit anti-alpha Tubulin monoclonal antibody ab52866 (Abcam) diluted 1000x for PrecisA Monoclonals.
For Triple A Polyclonals
- Alexa® Fluor 488 goat anti-rabbit IgG (ThermoFisher Scientific, product no: A11034) diluted 400x.
- Alexa® Fluor 594 goat anti-mouse IgG (ThermoFisher Scientific, product no: A11032) diluted 400x.
For PrecisA Monoclonals
- Alexa® Fluor Plus 488 goat anti-mouse IgG (ThermoFisher Scientific, product no: A32723) diluted 800x.
- Alexa® Fluor 594 goat anti-rabbit IgG (ThermoFisher Scientific, product no: A11037) diluted 400x.
NOTE: The dilution of the antibodies is to be considered as a guideline only. Optimal dilution must be determined by the user.