ICC Unfixed Cells—Direct Method
Note: This protocol is intended for use with the specific products mentioned within it. Substituting different products is not recommended.
Staining using a fluorophore-conjugated antibody
- Tris buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.4) or phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4)
(NOTE: for maximum signal intensity, we recommend using TBS with Invitrogen™ eFluor™ Nanocrystals)
- Fixation reagent: 4% formaldehyde in PBS (optional)
- Blocking reagent: IHC/ICC Blocking Buffer-Low Protein (cat. no. 00-4953) or High Protein (cat. no. 00-4952)
- Primary antibody: fluorophore-conjugated format
- Nuclear counterstain: DAPI or DRAQ5 (cat. no. 65-0880)
- Mounting medium: Fluoromount-G (cat. no. 00-4958) or Fluoromount-G with DAPI (cat. no. 00-4959)
- Humidified container in which to place the samples during incubations
- Glass coverslips
- Clear nail polish
- Cells are plated at an appropriate density and allowed to attach to the slide or dish (ex. 30,000 cells/chamber in an 8-chamber slide). Cells are usually plated one day prior to staining in order to achieve 60-80% confluency.
- Cover the cells with blocking solution (100 μL/chamber in an 8-chamber slide). To limit evaporation, use a piece of parafilm to tightly cover the opening of the chamber slide. Incubate in a humidified chamber for 30min at room temperature.
- Gently wash the cells 3 times in PBS or TBS (5 min/wash). Use a dropper to add PBS or TBS to the chamber followed by aspiration to remove the buffer.
- Dilute fluorophore-conjugated primary antibody or multiple fluorophore-conjugated antibodies, at manufacturer’s recommended dilution, in blocking reagent and overlay onto cells, protecting from light. Tightly cover the opening of the chamber slide with parafilm. Incubate in a humidified chamber for 1-2 hours at room temperature.
- Remove the parafilm cover and gently wash the cells 3 times in PBS or TBS (5 min/wash) as described in step 3.
- Optional: Fixation with 4% formaldehyde diluted in PBS for 15 minutes at room temperature. Wash cells 3 times in PBS or TBS
- Optional: Nuclei can be counterstained using DAPI or DRAQ5. It is necessary to select a counterstaining agent with a fluorescent emission spectra that does not overlap with the emission spectra of the other fluorophores used in the experiment.
- Mount and coverslip using Fluoromount-G or Fluoromount-G with DAPI. Seal the edge of the coverglass with clear nail polish.
- Allow slides to dry for 1-2 hours before visualizing.
- Slides can be stored at 4°C protected from light if needed.
For Research Use Only. Not for use in diagnostic procedures.