To obtain cells for immunofluorescence, 5–10 ml of subculture was grown to mid-exponential phase and fixed with 5% formaldehyde for 3–4 h at 30 °C. Cells were harvested at 1300×g for 5 min and washed once with 5 ml S-Buffer (50 mM HEPES pH 7.5, 1.2 M Sorbitol) before being resuspended in 1 ml S-Buffer. To induce spheroplast formation, 10 µl of 1 M DTT, 30 µl of glusulase, and 40 µl of 2.5 mg/ml zymolase were added to the cells and the mix was incubated for 30 min at 37 °C. The extent of spheroplasting was monitored under the microscope. The poly lysine (Lys) coated slides were prepared by adding 15 µl of 0.1% poly Lys per slide well and set aside for 15 min. Wells were washed three times with PBS. The fixed cells were centrifuged for 1 min at 5000 rpm and gently resuspended in 1 ml S-buffer. Following the addition of 0.1% Triton X-100, the mix was incubated for 5 min on rocker. The cells were centrifuged for 1 min at 5000 rpm and resuspended in 1 ml S-Buffer. To adhere the cells, 20 µl of cell suspension was added on to poly Lys-coated well and incubated it for 15 min. The cells were washed three times with PBS-T and the wells were blocked with 20 µl PBS/BSA for 5–10 min. Anti-HA antibody diluted 1:300 in 20 µl was added and incubated for 3–4 h or overnight in a humid chamber. The cells were washed four times in PBS-T. Anti-mouse IgG-Cy3 diluted 1:500 in PBS-T was added to the wells and incubated for 1–2 h. The cells were washed four times with PBS-T and once with only PBS. Overall, 20 µl of 1 mg/ml DAPI diluted 1:1000 in PBS was added to the wells and incubated for 5 min. The cells were washed four times with PBS. The cells were left to dry in the dark for 30 min at room temperature. Mounting medium was added and cells were viewed under the microscope.