Axio imager k z 2 microscope

Intracellular neutral lipids were stained by treating iRPEs with 2 μg/mL Nile red (Sigma-Aldrich, St. Louis, MO, USA) diluted in FMN medium for 30 min at 37 °C, as previously described [35 (link)]. Cells were washed 3× with PBS, fixed in 4% PFA for 10 min at RT, washed with PBS and mounted in mowiol. Fluorescence images were acquired with an Axio Imager K Z.2 microscope (Zeiss, Jena, Germany) and fluorescence intensities were calculated using ImageJ.

iRPE cells were grown on Matrigel-coated coverslips for 4 weeks. Cells were washed 2 times with PBS and fixed in 4% PFA for 15 min at RT, followed by blocking in 3% normal goat serum and 0.3% Triton x-100 in PBS for 1 h at RT. Primary antibody rabbit anti-ZO-1 (1:250, #40-2200c, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) diluted in blocking buffer was applied overnight at 4 °C. Afterwards, coverslips were washed 3 times in PBS and incubated with secondary antibodies conjugated to Cy3 fluorophore (Jackson ImmunoResearch, Cambridgeshire, UK) for 1 h at RT in the dark. Coverslips were washed 3 times in PBS, incubated with 0.8 μg/mL DAPI in PBS, washed 3 times in PBS, once in ddH₂O and mounted with mowiol. Immunofluorescence images were acquired using an Axio Imager K Z.2 microscope (Zeiss, Jena, Germany).

Porcine photoreceptor outer segments (POSs) were isolated as previously described and stored at −80 °C [57 (link)]. For phagocytosis assays, POSs were thawed on ice and washed twice with PBS and centrifuged at 3000× g. Around 10⁷ POSs were re-suspended in DMEM:F12 medium, supplemented with 10 μL FITC (1 mg/mL in DMSO; Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at 4 °C with mild agitation. The next day, POSs were washed twice with PBS, centrifuged at 3000× g and re-suspended in FMN medium. Ten POSs/cell were diluted in FMN medium and transferred into 24-wells with iRPEs grown on coverslips. After 4 h, excess POSs were removed by 3 washes with PBS. Cells were fixed in 4% PFA/PBS for 15 min at RT, followed by immunofluorescence staining for ZO-1 and nuclei counterstaining with DAPI. Fluorescence images were acquired using an Axio Imager K Z.2 microscope (Zeiss, Jena, Germany) and the numbers of bound and internalized POS particles and nuclei were quantified using ImageJ.