Ix81 fluorescent microscope system

The localization of PARK7, ZO-1, and the cytoskeletal actin architecture was investigated by immunofluorescence staining on frozen biopsy samples and FHs74Int cells. After repeated washing with PBS, slides were permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) for 15 minutes at RT, washed with Perm/Wash Buffer solution (BD Pharmingen), and incubated with a primary antibody specific for PARK7 (ab18257; rabbit, 1 : 1000, Abcam, Cambridge, US), ZO-1 (ab96587; rabbit, 1 : 1000, Abcam), or Alexa Fluor® 546 phalloidin (7.5 units/mL, A22283; Thermo Fisher Scientific) for 1 hour at RT. In case of PARK7 and ZO-1 staining, slides were incubated with antirabbit Alexa Fluor 568®-conjugated secondary antibody (1 : 1000, A11036; Thermo Fisher Scientific) or antirabbit Alexa Fluor 488®-conjugated secondary antibody (1 : 1000, A21206; Thermo Fisher Scientific) for 30 minutes at RT. Thereafter, the slides were washed with a Perm/Wash Buffer solution and coverslipped with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Sections were analyzed with an Olympus IX81 fluorescent microscope system.

Characterization of isolated HPMC as well as HPFs was performed by immunofluorescence staining. Cells were seeded in cell culture chambers (Sarstedt Kft., Budapest, Hungary) at a density of 104 cells/well and were incubated for 24 h at 37 °C. After washing with WashPerm solution, slides were permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) at room temperature for 15 min. Slides were incubated with primary antibody specific for α-SMA (mouse, 1:5000; Merck Life Science Kft. Budapest, Hungary) or CK-18 (mouse, 1:1000; Merck Life Science Kft. Budapest, Hungary) at room temperature for 1 h. Thereafter, the slides were washed and incubated with corresponding Alexa Fluor 488 conjugated secondary antibody (anti-mouse, 1:100; Thermo Fisher Scientific, Budapest, Hungary) at room temperature in the dark for 30 min. Finally, slides were coverslipped with ProLong Gold antifade reagent (Thermo Fisher Scientific, Budapest, Hungary). Appropriate controls were performed by omitting the primary antibodies to assure their specificity and to avoid autofluorescence. Sections were analyzed with an Olympus IX81 fluorescent microscope system (Olympus, Tokyo, Japan).