Ld c apochromat

For imaging of Wtf proteins expressed in S. cerevisiae (Figure 7D–F), we first grew 5 mL saturated overnight cultures in SC -His -Ura -Trp media. The next day, we diluted 1 mL of each saturated culture into 4 mL of fresh SC -His -Ura -Trp media. We then added β-estradiol to a final concentration of 500 nM to induce wtf expression and shook the cultures at 30°C for 4 hr prior to imaging.
Cells (5 µL concentrated culture) were then imaged on an LSM-780 (Zeiss) with a ×40 LD C-Apochromat (NA = 1.1) objective. A physical zoom of 8 was used which yielded an XY pixel size of 0.052 µm. The fluorescence of GFP was excited with the 488 nm laser and filtered through a 491–553 nm bandpass filter before being collected onto a GaAsP detector running in photon counting mode. The fluorescence of mCherry was excited with the 561 nm laser and filtered through a 562–624 nm bandpass filter before being collected onto the same detector.

HEK293A cells transfected with hHVRP1-EGFP or HVRP1*-EGFP in pNICE were grown on poly-D-lysine coated glass-bottom dishes (Mattek) for 1 to 2 days. Directly before imaging, live cells were washed in cold HBSS (Life Technologies) to remove residual FBS, and bathed in fresh HBSS. FM 4–64 dye (Life Technologies) was added to the solution (5 µM final concentration) to label the plasma membrane. Cells were imaged on a Zeiss LSM 780 confocal microscope with an LD C-Apochromat 63× immersion objective with 1.15 numeric aperture. For EGFP, excitation was at 488 nm, and emission band was 491–560 nm. For FM 4–64, excitation was at 561 nm, and emission band was 592–759. Primary cerebellar granule neurons growing on poly-D-lysine coated coverslips were fixated in 1∶1 methanol and acetone solution for ten minutes at −20°C. Immunocytochemistry was performed using either anti-HVRP1 antibody diluted 1∶1000, or anti-HVRP1 antibody diluted 1∶500 pre-incubated with peptide antigen (1∶500 dilution of a 1 µg/µl solution), and AlexaFluor-594-labeled goat anti-rabbit secondary antibody. Coverslips were then mounted onto Fisher SuperFrost Plus slides with ProLong Gold Antifade Reagent with DAPI (Life Technologies). For AlexaFluor 594, excitation was at 561 nm, and emission band was 592–759 nm. For DAPI, excitation was at 405 nm and emission band was 415–735 nm.

Confocal images were acquired at RT using a Zeiss LSM780 confocal microscope fitted on a Axiovert M200 inverted microscope equipped with a LD C Apochromat (40×, NA=1.1) water immersion objective (Carl Zeiss). Optical sections (512×512 pixels) were collected sequentially for each fluorochrome. Images of maps reconstructing the whole mounts were obtained at RT using a LD LCI Plan Apochromat (25×, NA=0.8) water & glycerol immersion objective (Carl Zeiss) or a Plan Apochromat (20×, NA=0.8) without immersion objective (Carl Zeiss). Optical sections (512×512 or 256×256 pixels, depending on map size) were collected sequentially for each fluorochrome. The generated data were processed and displayed using ZEN software. Maps were cropped if necessary to isolate the prostate epithelium from mesenchyme and surrounding tissue. Quantifications were performed manually using the scoring tool of ZEN software.

Transversal sections were imaged on a Zeiss Imager.M2 with an ApoTome.2 module using HXP 200C illumination. Immunostained sections of embryos of different stages were imaged with 40x Plan-Apochromat, 0.95 Korr (except Figures 5B and 20x Plan-Apochromat, N/A 0.8). Whole mount embryos were imaged on same microscope with 5x (EC Plan NeoFluar) or 10x (Plan-Apochromat, N/A 0.45) objective with Axiocam506 mono (fluorescence) and color (DIC/brightfield) cameras (except Figure 5B, imaged on Olympus MVX10 with Zeiss MRm camera). Figure 1B’ was imaged on Zeiss 710 inverted confocal microscope with LD C-Apochromat 40x/1.1 W Korr UV-VIS-IR objective.

Zebrafish embryos were mounted in 1% low-melting agarose supplemented with 0.2% (w/v) tricaine to stop heartbeats. The images were acquired using a Zeiss spinning disk confocal microscope system with CSU-X1 (Yokogawa) and ORCA-flash4.0 sCMOS camera (Hamamatsu) or a confocal microscope (LSM800, Zeiss) using an LD C-Apochromat × 40 (1.1 numerical aperture) objective. Imaris (Bitplane) was used for 3D image processing. Myofilament thickness was measured using the ZEN imaging software (Zeiss).

The 2D dataset provided with our notebooks was generated by plating U-251 glioma cells expressing endogenously tagged paxillin- GFP on fibronectin-coated polyacrylamide gels (stiffness 9.6 kPa)^{72 (link)}. Cells were then recorded live using a spinning-disk confocal microscope equipped with a long working distance of ×63 (NA 1.15 water, LD C-Apochromat) objective (Zeiss). The 3D dataset provided with our notebooks was generated by recording A2780 ovarian carcinoma cell, transiently expressing lifeact-RFP (to visualise the actin cytoskeleton), migration on fibroblast-generated cell-derived matrices^{81 (link)}. The cell-derived matrices were labelled using Alexa Fluor 488-recombinant fibronectin and the images acquired using a spinning-disk confocal microscope equipped with a 63x oil (NA 1.4 oil, Plan-Apochromat, M27 with DIC III Prism) objective (Zeiss). For both datasets, the spinning-disk confocal microscope used was a Marianas spinning-disk imaging system with a Yokogawa CSU-W1 scanning unit on an inverted Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 (Intelligent Imaging Innovations, Inc.). Images were acquired using a Photometrics Evolve, a back-illuminated EMCCD camera (512 × 512 pixels).