Vectashield liquid mounting media

CrFK cells were grown overnight on glass coverslips and infected with FCV at a multiplicity of infection (MOI) of 5 at the indicated times, or at an MOI of 10 for 30 min at 4 °C as indicated. The cells were treated with cytoskeleton buffer for 5 min and permeabilized in 4% formaldehyde solution for 5 min at room temperature (RT). The samples were washed three times with phosphate buffer saline (PBS) for 5 min, blocked with 0.5% gelatin in PBS for 40 min at RT, washed three times with PBS for 5 min, and incubated with the anti-FCV (FCV1-43, Santa Cruz Biotechnology, CA, USA) that recognizes an epitope on the capsid protein or the anti-JAM-1 (ab106114, Abcam, Cambridge, UK), at 4 °C overnight. Samples were washed three times with cold PBS for 5 min and incubated with the appropriate secondary antibodies (Invitrogen, MA, USA) for 1 h at RT. The samples were washed three times with PBS and incubated with 1 mg/mL of 4′6′-diamidino-2-phenylindole (DAPI) for 2 min. The samples were washed six times with PBS and three times with distilled water. The samples were treated with VECTASHIELD liquid mounting media (Vector Laboratories A.C., CA, USA) and analyzed using a Zeiss LSM-700 confocal microscope.