Tissue tek oct

The tissue sample was cut into 3–4 mm thick segments (Figure 1A) and placed in Optimal Cutting Temperature (OCT) compound (Tissue-Tek OCT, TED Pella Inc, CA, USA). Following that, the tissue samples were frozen using liquid Nitrogen. For staining, 8 μm thick slices were sectioned using a cryostat (Leica CM1850). Hematoxylin & Eosin (H & E, Figure 1B), Oil Red O and Masson's Trichrome Staining were used to define the fibrous tissue, lipids, calcification, and hemorrhage. Lipids were stained red in Oil Red O stain, and hemorrhage was identified both in H & E and Masson's Trichrome staining in dark pink color. Calcium deposits were lost in the sample preparation process but could be easily defined from both H & E and Oil Red O staining as they clearly show the boundary of calcification in dark purple color.

Adherent cells were fixed on ice in 4% PFA in 0.1 M PB-5% sucrose for 5 minutes; floating OVs and RCs were fixed for 25 minutes. On ice, RCs were immersed sequentially in 6.75% and 12.5% sucrose-PBS for 1 hour each, 25% sucrose-PBS overnight, 1 hour in a 2:1 ratio of 25% sucrose-PBS/OCT Tissue-Tek (Ted Pella), and snap-frozen on dry ice. 8 μm sections were mounted onto Superfrost Plus slides (ThermoFisher) and incubated overnight in primary antibodies in 2% normal donkey serum (NDS) and 0.1–0.2% Triton X-100 in PBS. Secondary antibodies were anti-mouse, -sheep and -rabbit IgG’s (H + L) coupled to Alexafluor-488, −546, or −647 (Invitrogen, 1:1,000). 10 μg/ml Hoechst 3342 (Molecular Probes) was used to visualize cell nuclei. Sections processed without primary antibody were used as controls. For whole-mount IHC, 3D RCs (Fig. 4) were blocked and permeabilized for 1 hour in 10% NDS, 0.2% Triton X-100 (TX100) in PBS, then incubated with primary antibodies against the visual pigments in PBS containing 2% NDS and 0.2% TX100, rinsed in PBS, and visualized with secondary antibodies (as above).