Ab6668

Isolated fixed retinas were permeabilized for 30 min in 0.5% Triton X-100 in PBS at RT. Retinas were then blocked in 10% goat serum, 1% bovine serum albumin, and 0.5% Triton X-100 in PBS for one hour at RT. Subsequently, retinas were incubated in goat anti-mouse F(ab) fragment (1:2000;ab6668, Abcam, Cambridge, UK) for one hour at RT. Mouse monoclonal anti-Brn3a (1:100; SC-8429, Santa Cruz Biotechnology, Dallas, TX) was diluted in blocking buffer and incubated on retinas overnight at 4 °C. Fluorescent-conjugated secondary antibody (1:500; Alexa Fluor 647, A21235, Invitrogen, Carlsbad, CA) was diluted in blocking buffer and incubated on retinas for two hours at RT. Bisbenzimide (1:100; Invitrogen H3569, Carlsbad, CA) was diluted in wash buffer and added to retinas for 20 min at RT. Four incisions were made in each retina cup to allow them to lie flat on a slide, and retinas were then mounted with Fluoromount-G (0100–01, SouthernBiotech, Birmingham, AL).

P10 retinas were dissected as described above and maintained at 33C in an incubator in oxygenated ACSF; 50 μM PregS or vehicle control was added to the ACSF for 30 min followed by washout 3× with oxygenated ACSF, and then incubation in oxygenated ACSF for 1.5 h for all conditions. The retina pieces were then fixed for 30 min in 4% PFA in PBS, followed by wash in 0.1% PBST. To block mouse endogenous antibodies, we incubated overnight in F(ab) fragment anti-mouse IgG (H + L) 0.1 mg/ml (ab6668; Abcam; 1:10 dilution) and 1:1000 rat anti-mouse monoclonal CD16/CD32 antibody (UCSF MAB core) prepared in 0.1% PBST. Retina pieces were again washed 3× in PBST followed by a 4 h incubation in blocking buffer: 5% donkey serum, 0.2% sodium azide, 0.1% Triton X-100. Anti-c-Fos antibody [2H2] (ab208942) 1:1000 was incubated overnight in blocking buffer. Retina pieces were washed 3× and then incubated in goat anti-mouse Alexa Fluor 488 (1:1000) overnight. Retina pieces were briefly washed in water and then coverslipped using Fluoromount with DAPI and imaged on a Zeiss 780 confocal microscope with a 63× oil immersion objective (NA 1.4).

Histological sections were immunohistochemically stained to visualise the MRP1 expression and to study the proliferative state of the tumours. Microtome sectioned tissue specimens of 5 µm were dehydrated at 50 °C overnight, followed by deparaffinisation and rehydration. Rehydrated slides were first incubated in sodium citrate buffer pH 6 at 95 °C for 30 min for antigen retrieval and cooled down to room temperature. For ki67 staining, slides were additionally incubated with 0.1% Triton X-100 in PBS for 10 min to permeablise the nuclear envelope. Slides were then incubated in 1% H₂O₂ for 15 min to quench endogenous peroxidase activity, followed by serum blocking for 1 h. Goat anti-mouse IgG Fab (Abcam, ab6668) was added for 1 h to block endogenous IgG. After washing in PBST, slides were incubated with primary against ki67 (Abcam, ab8191) or primary against MRP1 (Santa Cruz, sc-18835) overnight in a moisture chamber at 4 °C. Slides were then incubated with biotinylated mouse-rabbit polyvalent secondary (Abcam, ab64264), followed by streptavidin peroxidase (Abcam, ab64264) incubation. After washing, slides were developed using 3,3′-diaminobenzidine (DAB) substrate for 15 min then counterstained with Mayer’s haematoxylin QS (Vector, H-3404), and eventually mounted in coverslips with DPX.