Superfrost glass slide

To assess Elovl5 expression in brain slices mice were anesthetized using a cocktail of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) via intraperitoneal injection. The mice were intracardially perfused initially with a physiological solution (NaCl 0.9%) and then with 4% paraformaldehyde in 0.12 M phosphate buffer, pH = 7.2–7.4. Following perfusion, the brain and the spinal cord were removed and stored at 4°C for 24 h immersed in the same fixative and later transferred to a solution made of 30% sucrose in 0.12 M phosphate buffer for few days. Perfused brains and spinal cords were embedded in paraffin, cut into 5 μm-thick sagittal slices and mounted on superfrost glass slides (Fisher Scientific, Göteborg, Sweden) with Tris-glycerol supplemented with 10% Mowiol (Calbiochem, LaJolla, CA, United States).
To assess Elovl5 expression in glial cells in vitro, postnatal day 2 (P2) pups were cryoanesthetized in melting ice. The experimental plan was designed according to the guidelines of the NIH, the European Communities Council (2010/63/EU) and the Italian Law for Care and Use of Experimental Animals (DL26/2014). The study was conducted according to the ARRIVE guidelines.

After enucleation, eyes were hemisected equatorially and the gel vitreous was removed from the posterior eyecup. Samples were fixed for 30 min at 20°C in 4% paraformaldehyde plus 3% sucrose in 0.1 M phosphate buffer, pH 7.4, as described in Diaz et al. (2016) (link). Fixed samples were washed three times in phosphate-buffered saline (PBS; 0.05 M phosphate buffer, 195 mM NaCl, pH 7.4), cryo-protected in PBS plus 30% sucrose, soaked in embedding medium (O.C.T. Compound, Tissue-Tek) for 10 min, and freeze-mounted onto aluminium sectioning blocks. Transverse sections nominally 14 μm thick were cut consistently from the posterior pole of the eye, near the dorsal portion of the pecten, and thaw-mounted on Super-Frost glass slides (Fisher Scientific). Sections were air-dried and stored at −20°C until use.

IHC was performed on retina and ON cryo-sections and SC paraffin sections to validate protein expression of Rbpms (RGC survival, n=3–7) and NRN1 and GFP (AAV2-induced overexpression, n=4). Whole eyes with ONs were harvested and fixed in 4% PFA for 2 h at room temperature. After fixation, the tissue was placed in 20% sucrose overnight at 4 °C and embedded in optimum cutting temperature the next day. Sections (10 μm) were cut using a cryostat (Leica Biosystems, Richmond, IL, USA). Cross-sections of retina were transferred to Superfrost glass slides (Fisher Scientific). Slides were incubated in PBS for 10 min and blocked with SuperBlock Blocking Buffer at room temperature for 1 h. Primary antibodies (Supplementary Table S1) were diluted in SuperBlock. Each slide was incubated with the respective primary antibody and incubated overnight at 4 °C. Sections were then washed and incubated with AlexaFluor secondary antibody (Supplementary Table S1) for 1 h at room temperature. After rinsing, slides were mounted with ProLong Gold anti-fade reagent with DAPI. Sections were observed and captured using a Nikon Eclipse Ti-U Microscope containing the Nuance Multispectral imaging system and analyzed using Adobe Photoshop CS5 software.

Whole pancreata were dissected, weighed, washed in cold PBS and incubated for 24 hr at 4°C in HistoFix (CarlRoth). For β cell mass quantification, the pancreata were fixed in homemade cylindrical tube for form standardization of downstream quantification analysis. Each pancreas was then washed twice in PBS for 30 min each at room temperature, and dehydrated in 70% EtOH/dH₂O overnight, 95% EtOH/dH₂O for 3 hr and 100% EtOH for 3 hr at RT before being cleared with xylene for 15 min and embedded in paraffin. 5 μm sections were collected on SuperFrost glass slides (Fisher).