C apochromat

The 4^th MG was collected, embedded in optimal cutting temperature compound (OCT; Tissue-Tek cat.4583), and snap-frozen with liquid nitrogen. 6–8-µm sections were cut using a cryomicrotome (Leica). The sections were fixed with ice-cold acetone (Merck, Cat. 1.00014.2500), blocked with 5% normal goat serum (Jackson ImmunoResearch, Cat. 055-000-121), and stained with the antibodies listed in the Supplementary Table 1 for 30 min at room temperature. After washing with PBS (Gibco, cat. 18912-014) the sections were mounted with ProLong Gold Antifade Mountant with DAPI (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific, cat. 62249), or the nuclei were stained with Hoechst (Thermo Fisher Scientific, Cat. 62249), and then mounted with ProLong Gold Antifade Mountant without DAPI (Thermo Fisher Scientific, cat. P36930). Images were acquired using a spinning disk confocal microscope (Intelligent Imaging Innovations) with a Plan-Apochromat 20 × /0.8 or 10 × /0.45 objective or LSM780 confocal microscope (Zeiss) with C-Apochromat 40 × /1.2 or C-Apochromat 63 × /1.2 oil objective. Background subtractions, linear brightness and contrast adjustments, and median filtering for reduction of noise were performed with ImageJ software.

Micrographs were captured using a cLSM 880 (Carl Zeiss, Oberkochen, Germany) using 10×, 20×, 40× or 63× water immersion objectives (C-Apochromat, NA: 1.2, Carl Zeiss). Where Airyscan is noted, images were obtained using the Airyscan detector and mode on the cLSM 880 (Huff, 2015 (link)). Where linear unmixing is noted, images were obtained using linear unmixing mode in ZEN software (Carl Zeiss⁷). Z-stack maximum intensity projection images were obtained at 1 μm intervals for whole antennal overviews and 0.5 μm intervals for detailed sections and close-ups. All confocal images were adjusted for contrast and brightness with ZEN software (Carl Zeiss).

Cells were fixed with 4% formaldehyde, permeabilised with 0.2% Triton X-100 for 5 min, blocked with 5% BSA-PBS for 1 hr at room temperature, and incubated with anti-p-MLC2 (p-MLC2S19, 1:200 in 5% BSA-PBS) overnight at 4°C. Alexa-488 anti-rabbit secondary antibody (Life Technologies) was used at 1:500 for 1 hr at room temperature. F-actin was detected with Phalloidin (1 hr RT) and nuclei were stained with Hoechst 33258 (Life Technologies). Imaging was carried out on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss) with C-Apochromat × 40/1.2 NA (water) or a Plan Apochromat × 63/1.4 NA (oil) objective lenses and Zen software (Carl Zeiss). Line scan analysis was performed in ImageJ.

Immunohistochemistry was performed as previously described^{12 (link), 27 (link), 41 (link)}. Briefly, brains were fixed in 4% paraformaldehyde at E16.5 and coronally sectioned with a cryostat (Leica Microsystems) at a thickness of 60 μm. The slices were incubated with primary antibodies diluted in PBS containing 1% bovine serum albumin (BSA) and 0.01% Triton X-100 at 4 °C overnight followed by Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies diluted in PBS containing 1% BSA and 0.01% Triton X-100 for 1 h at room temperature. The nuclei were visualized by staining with Hoechst 33342 (Invitrogen). Confocal images were recorded using an LSM 780 built around an Axio Observer Z1 with Plan-Apochromat 20 × (numerical aperture (NA) 0.75), C-Apochromat 40 × (NA 1.2), or Plan Apochromat 63 × (NA 1.40) lenses under the control of LSM software (Carl Zeiss). Coronal sections of cerebral cortices containing the labeled cells were classified into CP and IZ, which were outer layer and middle layer, respectively. The number of all labeled cells in each region was calculated. To evaluate the morphology of the migrating neurons, projection images of EGFP-positive neurons were obtained from Z-series confocal images using LSM software. At least three independent fetal brains were electroporated and analyzed for each experiment. All experiments were performed in a blinded manner.

Fluorescence quantification for AtRGS1-YFP internalization was performed as described by Urano et al. (2012) (link) and Fu et al. (2014) (link). Sterilized, stratified seeds were germinated in 6-well plates containing 2 ml 1/2 × MS liquid medium (pH adjusted to 5.75 with 5 N KOH) at 23°C under darkness. Seedlings (7-day-old) were treated with 0 or 3% D-glucose (w/v) for 30 min. Hypocotyl epidermal cells located 2–4 mm below the cotyledon were imaged (Z stacks obtained) using a Zeiss LSM710 confocal laser scanning microscope equipped with a 20× Plan-NeoFluor (N.A. = 0.5) objective and a 40× C-Apochromat (N.A. = 1.20) water immersion objective. YFP fluorescence was excited by a 514 nm argon laser and detected at 526–569 nm by a photomultiplier detector. At least 10 sets of images from 5 seedlings were obtained for internalization quantification analysis by ImageJ software.

Fluorescence quantification for AtRGS1-YFP internalization was performed as described by Urano et al. [48 (link)] and Fu et al. [49 (link)]. WT, atg2, and atg5 seedlings (7 days old) were treated with 0% or 6% D-glucose (w/v) for 30 min. Root epidermal cells located 2–4 mm below the cotyledon were imaged (Z stacks obtained) using a Zeiss LSM710 confocal laser scanning microscope equipped with a 20 × Plan-NeoFluor numerical aperture (N.A. = 0.5) objective and a 40 × C-Apochromat (N.A. = 1.2) water immersion objective. YFP fluorescence was excited by a 514 nm argon laser and detected at 526–569 nm by a photomultiplier detector. At least 10 sets of images from seven seedlings were obtained for internalization quantification analysis by ImageJ software.