Alexa fluor 488 and 594 tagged secondary antibodies

Cells cultured on coverslips were washed with PBS and fixed with 4% PFA at RT for 10 min. Alternatively, for co-staining of CD44 and RPN2, cells were washed with PBS and fixed with ice-cold MeOH at −30°C for 10 min. After fixation, cells were washed 3 times with PBS and permeabilized with 0.05% Triton X-100/PBS at RT for 5 min. Cells were washed 3 times with PBS and blocked in blocking buffer (10% FBS/PBS) at RT for 20 min. Then the cells were incubated in the blocking buffer containing 1:200 diluted antibody against CD44 (Proteintech; cat. 15675–1-AP), ERp57 (SantaCruz; cat. sc-23886), HSP47 (SantaCruz; cat. sc-5293), RPN2 (SantaCruz; cat. sc-166421), mtTFA (SantaCruz; cat. sc-376672), GFP (MBL; cat. 598) and 58K Golgi protein (Abcam; cat. ab27043) at 4°C for overnight. The cells were washed 3 times with PBS and incubated in the blocking buffer containing 1:1000 diluted Alexa Fluor 488- and 594-tagged secondary antibodies (Thermo Fisher Scientific; cat. A-11001 and A-110037) at RT for 1 h. The cells were washed 3 times with PBS and mounted in ProLong Gold mounting medium containing DAPI (Thermo Fisher Scientific; cat. P36941). Confocal fluorescent images were recorded using LSM800 confocal microscope (Zeiss).