Dab freebase

HeLa cells were grown on glass bottom petri dishes (MatTek) to 70% confluency and transfected with pJJS243 or pMX0167. After 36 hours fixed at room temperature in 2% glutaraldehyde (EM grade, Agar Scientific) in 0.1 M cacodylate buffer pH 7.4 (CB) for 30-60 min on ice. Cells rinsed five times with CB for two minutes, blocked in 50 mM glycine in CB for 5 min on ice, and rinsed in CB again. For APEX-catalysed oxidation of 3,3-diaminobenzidine (DAB), freshly prepared 0.5 mg/ml of DAB freebase (Sigma) in HCL and 0.03 % H₂O₂ (10 mM; Sigma) was added and upon a brown precipitate (~15 minutes) the reaction halted by rinsing with CB fives times. Samples were post-fixed with 1% OsO₄ in CB for one hour in the dark and on ice, rinsed 5x ten minutes in chilled deionised water, dehydrated in a chilled ascending ethanol series (30%, 50%, 70%, 90%, 100%) with 100% ethanol at room temperature, then infiltrated with CY212 epoxy resin and polymerised. Areas containing dark granules were excised, mounted onto dummy resin blocks and sectioned using a Leica ultramicrotome parallel to the substratum. Pale gold 70 nm sections were contrasted with saturated aqueous uranyl acetate and Reynold’s lead citrate. Electron micrographs were recorded at 80kV on an FEI Technai Spirit TEM. The distance between mitochondrial membranes was measured using the segmented lines tool in ImageJ.

At least three weeks after intravitreal injection of AAV2-EF1α-DIO-miniSOG-f and AAV2-EF1α-DIO-tdTomato into Opn4^Cre mice, the mice were anesthetized with ketamine/xylazine and transcardially perfused with Tyrode’s solution followed by 4% formaldehyde / 0.1% glutaraldehyde in 0.1M phosphate buffered saline (PBS). The retina and brain were dissected and post-fixed with 4% formaldehyde in 0.1 M PBS on ice for 2h and then the brain was cut into 100-μm-thick slices. For miniSOG photooxidation, tdTomato expressed ipRGCs and ipRGC neurites were identified using a Leica SPE II inverted confocal microscope and the retina and brain tissue were fixed with 2.5% glutaraldehyde, 2.5 mM CaCl2 in 0.15 M Sodium Cacodylate buffer (CB) pH 7.4, and the tissue was rinsed with ice-cold CB, and blocked for 30 min with 50 mM glycine, 10 mM Potassium cyanide and 5 mM aminotriazole in CB. Freshly prepared diaminobenzidine (DAB free base, Sigma) in CB was added to the tissue, and ipRGCs and axons were illuminated with 450–490 nm light from a Xenon lamp for 20–25 min until a light brown reaction product was observed in place of the green fluorescence of MiniSOG. The tissue was then processed for SBEM. Although the method was optimized in several mice, all volumes presented in this manuscript were from one mouse.

Cells grown on glass bottom petri dishes (MatTek) were fixed at room temperature with 2% glutaraldehyde (EM grade, Agar Scientific) in 0.1 M cacodylate buffer pH 7.4 (CB). Cells were then incubated on ice for 30-60 min, rinsed twice with chilled CB, and blocked with 50 mM glycine in CB on ice for 10 minutes. To start the APEX-catalysed oxidation of 3,3-diaminobenzidine (DAB), a freshly prepared solution of 0.5 mg/mL DAB free base (Sigma) in HCL and 0.03% (10 mM; Sigma) H₂O₂ were added to the cells. Formation of reaction product (a brownish precipitate) was monitored by light microscopy. After 15 minutes, the reaction was halted by washing the cells twice with chilled CB. Samples were post-fixed with 1% osmium tetroxide in CB on ice for 1 hour, washed five times with distilled water, and dehydrated in an ascending ethanol series (30%, 50%, 70%, 90%, 100%) at room temperature. The dehydrated samples were infiltrated and embedded in CY212 resin. Areas containing brown precipitates were excised and mounted on dummy blocks and sectioned parallel to the substratum. Pale gold, 70 nm sections were contrasted with saturated aqueous uranyl acetate and Reynolds lead citrate. Electron micrographs were recorded at 80 kV or 120 kV on a FEI Tecnai Spirit TEM.