Ni e a1 hd25 confocal microscope

Slides with cryosections were dried up at room temperature and then boiled in the boiling buffer (800 ml milli-Q water, 4 ml 1 M Tris pH8, 1.6 ml 0.5 EDTA) for antigen recovery. Then the brain sections were blocked in 150 μl blocking buffer (0.1 M PBS; 10% NGS and 0.1% Tween 20) and incubated overnight with the following primary antibodies: 1:300 Anti-Nras, Abcam, ab77392; 1/500 Anti-GFP, Proteintech, 50430-2-AP; 1/500 Anti-mCherry, Abclonal, AE002. Signals were visualized using the 1:400 diluted secondary antibody conjugated with AlexaFluor-488, -561 or −647 (Proteintech and Abclonal). After nucleic DNA staining by 4′,6-diamidino-2-phenylindole (DAPI, Sigma), slices were mounted with fluorescent anti-fade mounting medium (Dako). Images were acquired using the Ni-E A1 HD25 confocal microscope (Nikon).

For in situ hybridization, following 4% PFA perfusion, the brains were post-fixed with 4% PFA overnight, dehydrated in 30% (w/v) sucrose until they sunk to the bottom of tubes. Dehydrated tissues were then embedded in O.C.T (SAKURA) and sectioned (20 μm) by a cryostat (Leica). Frozen brain sections were air dried at room temperature under RNA free hood followed by overnight hybridization with digoxigenin-labelled miR-29a-3p probe (miRCURY LNA, QIAGEN, 339112YD00616795-BEF see Table S1) at 59 °C in the RNase-free incubator. Afterwards, the brain slices were washed and incubated overnight with 1:1500 anti-DIG-AP (Roche) antibody followed by BM-Purple (Roche) staining according to a standard procedure. Images were checked under bright field microscope. For co-localization studies immunofluorescent staining for GFP and mCherry was performed as described above and the images were acquired using the Ni-E A1 HD25 confocal microscope (Nikon).

Transiently transformed tobacco leaves (lower epidermis) and rice protoplasts were observed with a Ni-E A1 HD25 confocal microscope (Nikon, Japan). Prior to image collection, the background auto-fluorescence was eliminated using untransformed tobacco leaves or rice protoplasts. The GFP fluorescence signal was exited at 488 nm and emission was collected at 500–550 nm. The chlorophyll auto-fluorescence was exited with 561 nm laser and emission was collected at 600–700 nm. For NaCl treatments, protoplasts were incubated in W5 solution with 250 mM NaCl, or 50 μM E-64d, or both NaCl and E-64d, for 30 min at room temperature. For inhibitor treatments, transformed protoplasts were incubated in W5 solution containing 200 nM AZD8055, or 1 μM ConA, or both AZD8055 and ConA, for 3 h at room temperature.