Sea blocker

Eyes from Pax6-SPP^-/- and WT control mice at around two months of age were harvested, frozen in OCT compound, and stored at -80°C until further processing. Frozen tissue was sectioned using a Cryostar NX70 (Thermo Scientific, Rockford, IL). Tissue sections were fixed with 4% paraformaldehyde, washed with 1× phosphate-buffered saline (PBS), and then permeabilized with 0.3% Triton X-100 in PBS. The slides were blocked using 1× sea blocker (Thermo Scientific) plus 5% horse serum for 1 hr at 25°C, then incubated with anti-SPP antibody (Bethyl Laboratory, A304-404A, Waltham, MA) in blocking buffer at 4°C overnight. Slides were washed three times with 1XPBS and incubated with Biotin-conjugated secondary antibody for 2 hr at 25°C. Slides were then washed four times with 1xPBS and developed using a Vector VIP substrate kit according to company instruction (Vector Laboratories, Burlingame, CA. Cat# SK-4600). Vector Methyl green was used for counter staining. Slides were mounted with Permount mounting medium (Thermo Scientific, SP15-100) and image acquisition and data analysis were performed using a Leica DM4000 microscope (Leica Microsystems, Buffalo Grove, IL). SPP staining was quantified using Image J software.

Vero cells were seeded in LabTek 4-chamber slides (Corning, Big Flats, NY) and infected with VC1 virus (1 PFU/cell for 24 hr) and dTat-gK4 or dTat control peptide were added to culture medium at a concentration of 20 μg/ml. Cells were fixed with 4% paraformaldehyde, washed with 1× PBS, and then permeabilized with 0.3% Triton X-100 in PBS. The cells were then blocked using 1× sea blocker (Thermo Scientific, Rockford, IL) for 1 hr at 25°C, washed 2 times with 1× PBS, and incubated with anti-V5 (Bethyl Laboratories, Montgomery, TX; catalog number A190-119A) to detect gK and anti-GM130 (Abcam, Cambridge, MA; catalog number ab52649) antibodies. Cells were then washed three times in PBS, and incubated with chicken anti-goat AlexaFluor 488 (for gK) (Life Technologies, Carlsbad, CA) and chicken anti-rabbit AlexaFluor 594 (for GM130) (Life Technologies, Carlsbad, CA) for 2 hr at 25°C. Slides were washed 3 times with 1x PBS and mounted with Prolong Gold (Invitrogen, Carlsbad, CA). The fluorophores were imaged in separate channels by confocal microscopy using a Leica SP5-X confocal microscope, image acquisition, and data analysis system (Leica Microsystems, Buffalo Grove, IL).