Plan apo 63

Images shown in all figures are representative of data acquired from at least N = 3 animals per experiment. Animals under deep isoflurane anesthesia were perfused transcardially with ice-cold PBS, followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). Before sectioning, tissues were cryoprotected by immersion overnight in 15% sucrose, followed by 24 h in 30% sucrose in PBS. Tissues were then frozen in OCT compound (Fisher) using a beaker of 2-methylbutane chilled in dry ice. Ten- or 50-µm-thick cryosections were stored at −80°C until use. Whole brains and hearts were imaged immediately after dissection using an Olympus SZX-12 with an SZX-RFL2 coaxial fluorescence attachment. Cryosections were imaged using a Leica TCS SP8X confocal microscope equipped with LAS X software and a 63× oil immersion objective (1.4 N/A). Some images were obtained using a Zeiss Axiovert 200 inverted microscope equipped with AxioVision software and a Plan-Neo 100 Å∼/1.30 and Plan-Apo 63 Å∼/1.40 oil-immersion objectives (Immersol 518F; Carl Zeiss, Inc.) or a Plan-Neofluor 20× dry objective.

For nuclear microtubule analysis, an ROI was drawn in Fiji over the nucleus. Fiji was used to threshold α-tubulin signal, and the area of the signal was measured within the ROI. This was performed on 8 cells per field of view, with 3–6 fields per condition per experiment. For high-resolution microtubule analysis, high-resolution confocal images were acquired with a Zeiss 880 Confocal with AiryScan FAST microscope with GaAsP detector and camera (Zeiss) using a Plan-Apo 63× oil-immersion objective (NA 1.40) and 488 and 561 nm lasers. Imaging was performed at RT, and images were acquired with the Zeiss 880 software and with the AiryScan detector. Images were then processed with AiryScan. All image analysis, including Z-stack compression, was performed in Fiji. For microtubule density, an ROI was drawn at the MTOC and the cell periphery. Fiji was used to threshold α-tubulin signal, and the area of the signal was measured within the ROI. This was performed on at least 10 cells per condition per experiment. For junction analysis, the number of contacts between α-tubulin and VE-cadherin was counted and normalized to the junction length. At least 15 junctions were measured per condition per experiment.

Cells were seeded on glass coverslips in 24 well plates. Cells were fixed in 4% PFA for 10 min and subsequently permeabilized for 5 min with 0.5% X-100 Triton. Cells were blocked in 0.5% skin fish gelatine for 1 h. Antibodies were diluted in the blocking solution. Primary antibodies were goat anti-lamin B1 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse anti-lamin A/C (4C11) Active Motif (Carlsbad, CA, USA). Secondary antibodies used were donkey anti-mouse and donkey anti-goat conjugated to Alexa Fluor 488 and Alexa Fluor 647 (Invitrogen, Waltham, MA, USA). Cytoplasmic loading was conducted by permeabilising live cells with a 750 μg/mL solution of saponin containing a secondary goat anti rabbit IgG Alexa Fluor 546 (80 μL stock solution per 1mL of detergent solution) at 4 °C for 5 min then fixed immediately with cold 4% PFA. Samples were imaged immediately. Fixed cells were imaged on the LSM5 Zeiss Inverted 510 META laser scanning microscope (Zeiss, Oberkochen, Germany) using a Plan Apo 63 × 1.4 NA oil immersion lens. Images were acquired using Zen2009 operating software (Zeiss). Samples of cytoplasmic loading and the hormone removal experiment were imaged on a Zeiss LSM880 Airyscan using a Plan Apo 40 × 1.3 NA oil immersion lens. Images were acquired and pre-processed using ZenBlack (Zeiss). Collected images were analysed in FIJI [30 (link)].