O4886

661 W cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes. Cells were permeabilized and blocked in PBS containing 4% BSA and 0.5% Triton X-100 for 1 hour at room temperature, incubated overnight with primary antibody at 4 °C, and then subjected to immunohistochemistry as previously described36 (link).
Primary antibodies were rabbit anti-opsin blue (1:500; chemicon, AB5407), rabbit anti-opsin red/green (1:500; chemicon, AB5405), rabbit anti-cone arrestin (1:500; Millipore, AB15282) and mouse anti-rhodopsin (1:10000; sigma, o4886).

Tissue sections were air‐dried for 30 min, rehydrated with PBS and incubated in blocking buffer at RT for 1 h. ICC blocking buffer composed of 0.1% Saponin, 1% BSA and 2% NGS or the appropriate serum depending on the secondary used. IHC blocking buffer composed of 2% NGS, 1% BSA and 0.05–1% Triton X‐100. Primary antibodies used in the range of 1:100–1:1,000. Primary antibodies used; Anti‐LAMP1 (rat monoclonal, 1:500, [1D4B] (ab25245), Abcam), anti‐Gat₁ (rabbit polyclonal, 1:1,000, K‐20, SC‐389 Santa Cruiz), anti‐Rhodopsin (mouse, 1:2,000, O4886, Sigma) and anti‐GFP, (goat polyclonal, 1:1,000, ab6673, Abcam). Sections were then incubated in primary antibody overnight at 4°C, washed with PBS and incubated with secondary antibody for 2 h at RT in blocking solution. Sections were then washed and counterstained with DAPI (4’,6‐Diamidino‐2‐Phenylindole, Dihydrochloride; 1:1,000 dilution in 1× PBS (Molecular ProbesTM, USA)). Subsequently, sections mounted under coverslips with fluorescence mounting medium (Dako, Denmark) and stored covered at 4°C to prevent exposure to light. Immunocytochemistry followed the same procedure as immunohistochemistry. Negative controls omitted the primary antibody.

POS fragments were isolated and purified from porcine eyes as described in [19 (link)]. The fragments were suspended in RPE medium containing 10% fetal bovine serum (FBS, Gibco). The POS-media were then added to the apical side of the cells cultured on inserts and incubated for 2 h at 37 °C with 5% CO₂. After this, the cells were washed with PBS, fixed, and immunostained as described above. POS particles were immunolabelled with primary antibody anti-opsin (1:1000, O4886, Sigma Aldrich) and actin filaments were stained with Phalloidin (1:800). 2–3 replicate inserts from 2–3 maturation experiments were used at both time points.
Z-stack images were acquired with laser scanning confocal microscope Zeiss LSM800 with 63x/1.4 oil immersion objective with interval of 100 nm. From each sample, five Z-stack images were captured from randomly selected areas. These images were resliced with ImageJ to 512 xz-slices which were converted to MIPs of 20 consecutive xz-slices, from which the internalized POS particles were then manually calculated. In addition, the overall number of cells was calculated to obtain the average number of POS particles per cell.