Proteinase k

At post-natal (P) day 5 rd1 animals were killed and the eyes rapidly enucleated in an aseptic environment. The entire eyes were incubated in R16 serum-free culture medium (Gibco, Carlsbad, CA), with 0.12% proteinase K (MP Biomedicals, Illkirch-Grafenstaden, France), at 37°C for 15 min, to allow preparation of retinal cultures with retinal pigment epithelium (RPE) attached. The proteinase K was inactivated with 10% FCS (Gibco) in R16 medium, and thereafter the eyes were dissected aseptically in a Petri dish containing fresh R16 medium. The anterior segment, lens, vitreous, sclera, and choroid were carefully removed by fine scissors, and the retina was cut perpendicular to its edges, giving a cloverleaf-like shape. Subsequently, the retina was transferred to a culture dish filter insert (COSTAR, NY) with the RPE layer facing the membrane. The insert was put into a six-well culture plate and incubated in R16 medium with supplements [18 (link)] at 37°C. The full volume of nutrient medium, 1 ml per dish, was replaced with fresh R16 medium every second day.
At P11, the cultures were incubated for 24 h with 6-Fluo-10-NAD⁺ at a concentration of 100 μM. At P12, the cultures were stopped by 45 min fixation in 4% PFA. This was followed by graded sucrose cryoprotection, embedding, and collecting of 12-μm-thick retinal cross-sections on a Thermo Scientific NX50 cryotome.

Mouse monoclonal anti-HO1 (SC-136960), rabbit polyclonal anti Nrf2 (SC-722), mouse monoclonal anti-p-JNK (SC-6254), mouse monoclonal anti-COX-2 (SC-514489), mouse monoclonal anti-p-NFκB (SC-271908), mouse monoclonal anti-Bcl2 (SC-7382), mouse monoclonal anti-caspase-3 (SC-56053), ABC Elite kit (SC-2018), Ultra Cruz mounting media (SC-516212), mouse anti-rabbit IgG-R (SC-2492), and 3,3’-diaminobenzidine peroxidase (DAB) (SC-216567) were purchased from Santa Cruz Biotechnology. The horseradish peroxidase-conjugated secondary antibodies were obtained from Abcam (ab-6789, ab-6721). p-NF-κB Elisa kit (Cat # SU-B28069) and Nrf2 Elisa kit (cat. no. SU-B30429) were purchased from (Shanghai Yuchun Biotechnology, China). HO-1 Elisa kit (cat. No. E-EL-R0488), and TNF-α Elisa kit (cat. No. E-EL-R0019) were got from Elabscience. PBS tablets and proteinase K were obtained from (MP Bio, United States). Formaldehyde, hydrogen peroxide (H₂O₂), reduced glutathione (GSH), trichloroacetic acid (TCA), 1-chlor-2,4-dinitrobenzene (CDNP), N-(1-naphthyl)ethylenediamine dihydrochloride, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), and carveol (catalog No: 192384 a mixture of isomers, with 97% purity) was purchased from (Sigma-Aldrich, United States). All-trans retinoic acid (ATRA) of the highest analytical grade (99% HPLC) was purchased from the local pharmaceutical industry (GSK).

5-10 mg of brain tissue was lysed using cell lysis solution (Qiagen) and Proteinase K (MP Biomedicals LLC) was added and incubated at 55°C over night. Samples were treated with RNAse A solution (Qiagen) and incubated at 37°C for 1 h. Protein precipitation solution (Qiagen) was added, and the samples were placed on ice for 5 min. 100% isopropanol was added to the supernatant and DNA was precipitated with 70% ethanol and hydrated in Tris EDTA buffer (TE-buffer) at 55°C for 5 min and stored at 4°C for SNP detection.

Interphase FISH was performed using a WWTR1-CAMTA1 fusion probe (BACs RP11-1120, RP11-980). The red signal (rhodamine) flanked the distal region of WWTR1 while the green signal (FITC) labeled the proximal region of the CAMTA1 gene.
3 μM sections were deparaffinized with xylene and dried with ethanol after baking at 56°C for 16 hours. All tissue sections were pre-treated with a 30% solution of pre-treatment powder in 2xSSC and digested for 10 minutes with Proteinase K according to the instructions of the suppliers (MP Biomedicals Illkirch, France). After a second dehydrating step, the probes were applied to the sections and the covered slides were sealed with rubber cement, heat-denatured and hybridized at 37°C for 16 hours. Subsequently, all sections were counterstained with DAPI I in mounting medium (1000 ng/ml, Abbott, Wiesbaden, Germany) and visualized under a Zeiss Axioplan microscope using a HBO103 lamp and the appropriate filters for the three fluorescent dyes. A negative control was used in each case. A case was interpreted as positive when at least 10 of 50 counted tumor cells (20%) showed a (yellow) fusion signal.

The organotypic retinal explant cultures were prepared as described previously [53 (link)]. At postnatal (P) day 5, rd1 animals were killed, and the eyes were rapidly enucleated in an aseptic environment. The entire eyes were incubated in R16 serum-free culture medium (Gibco, Carlsbad, CA) with 0.12% proteinase K (MP Biomedicals, Illkirch-Grafenstaden, France) at 37 °C for 15 min to allow for preparation of retinal cultures with retinal pigment epithelium (RPE) attached. proteinase K was inactivated with 10% FCS (Gibco) in R16 medium, and thereafter, the eyes were dissected aseptically in a Petri dish containing fresh R16 medium. The anterior segment, lens, vitreous, sclera, and choroid were carefully removed by fine scissors, and the retina was cut perpendicular to its edges, giving a cloverleaf-like shape. Subsequently, the retina was transferred to a culture dish with membrane insert (COSTAR, NY), with the RPE layer facing the membrane. The insert was put into a six-well culture plate and incubated in R16 medium with supplements at 37 °C. The full volume of nutrient medium, 1 mL per dish, was replaced with fresh R16 medium every second day. The procedures for retinal explant cultures derived from Rho ^P23H/+ animals were the same as for rd1 mice, except that the Rho mutants were killed at P14 and then cultured for four days until P18.