**5.3. Confocal laser scanning microscopy**

Human embryonic kidney cells (HEK 293 Cell line) were treated for 20 min with glutaraldehyde 2.5% in PBS, washed three times with PBS, permeabilized for 10 min with 0.1% Triton-X100 and finally washed in PBS. Cells were then blocked for 20 min with 1% BSA in PBS, and after apposite washes, cells were incubated with rabbit anti-EF1A1 antibody (GenScript, Piscataway, NJ, USA) and mouse anti-His polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:300 in 1% BSA for 1 h. After washing three times with PBS, cells were incubated for 1 h with the appropriate secondary antibodies conjugated to fluorochromes and diluted 1:1000 in 1% BSA. Incubation with TOPRO 3-Iodide (Invitrogen Molecular Probes Eugene, OR, USA) diluted 1/1000 in BSA 1% was done for staining of the nucleus. After this, cells were washed properly with PBS and then observed with a Nikon Confocal Microscope C1 equipped with an EZ-C1 Software for data acquisition by using 60× oil immersion objective.
