Immunocytochemistry (ICC) is classically defined as a procedure to detect antigens in cellular contexts using antibodies. Immunocytochemistry (ICC) is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes.
--Fluorophore-conjugated secondary antibody/antibodies
--Cells of interest
--Tissue culture medium appropriate for the cells
--6-well tissue culture plates
--Broad-Tipped Forceps, 4 1/2" long
--Coverglass staining jar
--PBS, pH 7.4 (8 mM Na2HPO4, 1.4 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, adjust pH to 7.4 with NaOH)
--Antigen Retrieval Buffer (100 mM Tris, 5% (w/v) urea, adjust pH to 9.5 with HCl)
--0.1% (v/v) Triton® X-100 in PBS
--DAPI nucleic acid stain, 1 mg/mL
--Nail polish or glue (e.g. Duco Cement)
1) Preparation of cell samples: Fix the cells in a suitable fixative, such as 4% paraformaldehyde, for 10-15 minutes. Permeabilize the cells by treating them with a permeabilizing agent, such as 0.1% Triton X-100, for 5-10 minutes.
2) Blocking: Block non-specific binding sites by incubating the cells with a blocking buffer, such as 5% BSA or 10% FBS, for 30 minutes to 1 hour at room temperature.
3) Primary antibody incubation: Dilute the primary antibody in the blocking buffer and incubate the cells with the primary antibody at 4°C overnight or at room temperature for 1-2 hours.
4) Wash: Wash the cells with a suitable buffer, such as PBS, to remove unbound primary antibody.
5) Secondary antibody incubation: Dilute the secondary antibody in the blocking buffer and incubate the cells with the secondary antibody at room temperature for 30 minutes to 1 hour.
6) Wash: Wash the cells with a suitable buffer, such as PBS, to remove unbound secondary antibody.
7) Counterstain (optional): Counterstain the cells with a nuclear stain, such as DAPI or Hoechst, to visualize the nuclei.
8) Mounting: Mount the cells using a suitable mounting medium, such as glycerol or mounting medium containing DAPI.
9) Imaging: Visualize the stained cells using a fluorescence microscope.
It is important to optimize each step of the protocol to achieve the best results, as different samples and antibodies may require different conditions for optimal staining.