Prolong gold mounting medium

For Hematoxylin and Eosin (H&E) staining, spleens were fixed overnight in formaldehyde/zinc fixative, paraffin embedded, sectioned and stained using a standard H&E protocol. For immunofluorescence, spleens were snap frozen on dry ice in OCT (Tissue-Tek) and stored at −80°C. 6-µm frozen sections were cut, fixed with 4% paraformaldehyde for 20 min, blocked with 10% FBS, and stained overnight at 4°C with either guinea pig antibody to LCMV (1:1,000 dilution), rabbit anti-mouse CD3 antibody (Abcam, 1:200), rat anti-mouse MOMA-1 antibody (Abcam, 1:200) or rat anti-ER-TR7 antibody (1:250, Abcam). Tissues were washed, incubated at 1 h with Alexa Fluor 488–conjugated antibody to guinea pig IgG (1:200, Invitrogen), Alexa Fluor 568-conjugated anti-rabbit IgG (1:200, Invitrogen) and Alexa Fluor 488- or 568-conjugated antibody to rat IgG (1:200, Invitrogen), washed, stained with 4,6-diamidino-2-phenylindole (DAPI) and mounted using Prolong Gold mounting medium (Life Technologies). Images were taken with a Zeiss Axiovert S100 immunofluorescence microscope fitted with an automated xy stage, an Axiocam color digital camera, and 5×, 10× and 20× objectives.

To induce CNV in C57BL/6J mice, laser-induced ruptures of Bruch’s membrane were performed with an argon laser photocoagulator (blue-green light) mounted on a slit-lamp (Space Coast Laser Inc.; Palm Bay, FL). Four lesions were created in both the left and right eyes of each mouse. Laser parameters used were 100-μm spot size, 0.1-second duration, and 0.1 Watts ^{24 (link)–27 (link)}. On day-3 post-laser treatment, mice were divided into two groups and received tail-vein injections of AS-VCAM-1 hAuNP or NS-hAuNP at a concentration of 0.5 mg/kg in PBS. Twenty-four hours after the probe was incorporated, the mice were sacrificed and the eyes were fixed in neutral buffer formalin (NBF) for 30 min. The anterior segments and lenses were removed while submerging the eye in NBF solution. The choroid-Bruch’s membrane-RPE complex was dissected as previously described ^{28 (link)–29 (link)} and transferred into tris-buffered saline (TBS) before immunostaining. Tissues were blocked/permeabilized in 10% donkey serum with 1% Triton X-100 and 0.05% Tween 20 in TBS for 2 hours and were then counter-stained for IB4 conjugated to Alexafluor-488 (Life Technologies; Grand Island, NY). The tissues were then mounted with Prolong Gold mounting medium (Life Technologies; Grand Island, NY). Images were taken using an epifluorescence Nikon Eclipse Ti-E inverted microscope (Melville, NY).

Chamber slides (Lab-Tek II) were seeded with 1 × 10⁴ cells in 1 ml medium and incubated up to 50–70% confluence. Cells treated with SHetA2 or an equivalent volume of DMSO were fixed in 2% paraformaldehyde in PBS, pH 7.4 for 30 min at room temperature, washed with PBS three times and then permeabilized with 0.5% Triton X-100 in PBS, pH 7.4 for 5 min. After three PBS washes, cells were incubated in PBS containing 0.1% BSA in PBS for 30 min at 37°C and then with p53 DO-1 antibody for 1 hr at 37°C. After three PBS washes, cells were incubated with the secondary antibody tagged with fluorochrome (Alexa fluor 488 or 647) for 1 hr at 37°C and washed thrice in PBS. Nuclei were stained with 300 nM DAPI for 7 min at room temperature and cells were washed thrice in PBS. Cells were covered in Prolong Gold mounting medium (Life Technology, Carlsbad, CA) and 1 mm thick cover glasses and left to dry overnight at room temperature and then imaged with a confocal microscope Leica SP2 (Leica, Wetzlar, Germany) at 63×.