The purpose of using BSA in washing solution is that BSA binds all potential binding sites so that too much non specific stuff does not bind to to real antigen which you are trying to find. When antibody is added to this solution in the next step, the antibody replace the BSA-antigen site and changes to Antibody-antigen bound site.

Now in the next step you want to remove non-specific binding of antigen, that is why again solution with BSA is used to get rid of non-specific binding and that replaced by BSA only at non-specific sites.

If you do not use BSA at all, your antibody will bind to specific and non-specific antigens and you will get hugh background.

This the cheapest method to detect antigen. Alternatives will be more expensives.

Good luck, Keep the good work

ICC is a simple and quick method to detect the antigen of your interest by following the user manual protocol and specific antibody. The ICC method is developed in order to target the specific protein of interest and to minimize the nonspecific binding of Ab with other antigenic sites in a experimental cell. Each step, material, dilution, number of washing and Ab dilution are standardized, peer reviewed and published before the methods are brought for the research. However, minor changes are made in the lab in order to optimize the physical conditions. The ICC has several advantages over other methods as it can be completed using few cells, changes of antigen at the level of cell and sub-cellular level can be analyzed.

I agree with Prof. Juneja that BSA blocks the nonspecific sites that otherwise give nonspecific binding with Ab. To avoid the nonspecific binding, strictly follow the ICC protocol and ensure that the materials including Ab are working fine in order to get the reliable results. Good luck

Hello Ipek, in the attachment you will find a protocol which I have sucessfully used. I shortly have explained the different working steps . I hope it will help you. Good luck!