Immunocytochemistry (ICC) is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes.
The antibodies bound on diffirent epitotoes can then be detected using several different methods.
Immunocytochemistry enables researchers to evaluate whether or not cells in a particular sample express the antigen in question.
In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.
In the present experiment we are identifying the neurons by the presence of neuron-specific marker Neurofilament-H in the primary cotical neuronal culture.
Adopted from BioRender
1. 4% Para formaldehyde in PBS
2. Triton X-100, Milli Q water
3. Bovine serum albumin (BSA)
4. 5% w/v BSA in Phosphate buffered saline (PBS)
5. 0.1% w/v BSA in PBS
6. Primary antibody produced in rabbit
7. Goat-Anti rabbit secondary antibodies.
8. Inverted florescent microscope
9. TMB substrate for localization(20X)
Procedure of Immunocytochemistry
1. Wash the cultures two times with PBS and then fix for 30 minutes with 4% paraformaldehyde – PBS at room temperature followed by permeabilisation with 0.2% Triton X-100 for 5 minutes.
2. Block the non-specific binding by incubating the cells with PBS containing 5% BSA w/v for 1 h at room temperature.
3. After blocking, wash once with PBS and incubate with primary antibody overnight at 4°C In the present experiment we are using specific antibody as neuronal marker.
The antibody should be diluted with PBS with 0.1% BSA (w/v) according to manufacterur’s instructions.
In the present case we are diluting 1part of antibody with 399 parts of 0.1% BSA-PBS.
4. Once the incubation time is over, remove the primary antibody and wash three times with 0.1% BSA-PBS
5. After washing incubate the cultures with secondary antibodies which was goat anti rabbit IgG conjugated with HRP (1:3000) in 0.1% BSA-PBS for 1 hour at room temperature.
6. Following the incubation, remove the antibody solution and wash three more times with 0.1% BSA-PBS.
7. Add 200μl of TMB substrate to each of the well and incubate in dark for few minutes.
Remove the substrate as soon as color starts appearing and observe under the microscope