Ez c1 acquisition software

Third instar larvae were dissected in ice-cold Ca²⁺-free HL3 solution (Stewart et al., 1994 (link)). HL3 was then replaced with PBS and larvae fixed for 10 min in PBS with 4% formaldehyde. Fixed preparations were mounted in Vectashield (Vector Laboratories), and images were collected using EZ-C1 acquisition software (Nikon) on a Nikon Eclipse C1si confocal microscope (Nikon Instruments, United Kingdom). Images were captured using a 40×/1.3 NA oil objective.

In some experiments, Trex1 was visualized using immunofluorescence staining from OCT sections of tumour tissue. Slides were fixed with 4% PFA and stained overnight for Trex1 (BD bioscience 611986 1:200 o/n). Imaging was performed on a Nikon Spectral C1 confocal microscope (Nikon C1si with EZC1 acquisition software, Nikon Instruments) with Plan Apo 10X/0.45 air, Plan Apo 20X/0.75 air, and Plan Apo 60X/1.40 oil objective lenses (Nikon). Some immunofluorescence imaging was performed on a Yokogawa CSU-W1 spinning disk confocal microscope with × 20 0.45 Plan Fluor objective (Nikon). All images were taken with a channel series. Images were analysed with Image J software for determination of CD31 or lectin area.

A Nikon Eclipse (Ti-E) inverted C1 confocal microscope (Obj 20×; 40×; 60×; 100×) equipped with two lasers and a motorized stage was used. The lasers mounted on the microscope are: an Argon ion (Spectra Physics, Mountain View, California) laser emitting fluorescence at 488 nm wavelength and He-Ne laser (Melles Griot Florence, Italy) emitting fluorescence at 543 nm wavelength.
For scanning along the axial (z) dimensions, a Plan Apo 60×, high numerical aperture (NA = 1.40) oil immersion objective (Nikon Florence, Italy) was chosen and a Z-stack of images was acquired at 0.15 μm increments, according to Nyquist criterion, automatically calculated by the Nikon EZ-C1 acquisition software. For 3D image reconstruction, the NIS Elements AR 4.30 software (Nikon Florence, Italy) was used.

Third instar larvae were dissected in chilled Ca²⁺-free HL3 solution (prefixation HL3; Stewart et al., 1994 (link)), and fixed for 10 min in PBS with 4% formaldehyde. Unless otherwise specified, visualization of fluorescent tags was performed via direct imaging without immunostaining. For immunostaining, the dissected preparations were permeabilized in PBS containing 0.1% Triton X-100 (PBT) at room temperature for 1 h. After permeabilization, samples were blocked in PBT with 4% bovine serum albumin for 30 min at room temperature, incubated with primary antibodies (Table S3) overnight at 4°C, and finally incubated with secondary antibodies (Table S3) for 2 h at room temperature. For myc and HA immunostaining, samples were permeabilized for 30 min with PBT, blocked for 1 h with 5% Normal Goat Serum in PBT, and incubated with anti-myc or anti-HA overnight at 4°C. Processed preparations were mounted in Vectashield, and images were collected using EZ-C1 acquisition software (Nikon) on a Nikon Eclipse C1si confocal microscope (Nikon Instruments) with 488 nm (for GFP and Alexa-488 signals), 561 nm (for tdTom, mCherry, and Alexa-594 signals) and 638 nm (for Alexa-647 signal) lasers. Images were captured using a 40×/1.3NA oil Nikon Plan Fluor DIC H/N2 infinity-0.17 WD 0.2 objective, or a 60×/1.4NA oil Plan Apo VC infinity-0.17 DIC N2.

Cells were seeded on glass slides, and fixed with Paraformaldehyde (PFA) 4% in PBS (phosphate-buffered saline); fixed cells were permeabilized with Triton™ X-100 (Sigma #T8787) 0.05% for 15’ at RT, and blocked for 1 h with 10% goat serum (Gibco #16210-072); Lamin B1 primary antibody (Santa Cruz #sc-30264) was incubated overnight at 4 °C; slides were washed 3X with PBS and incubated 1 h at RT with the appropriate Alexa Fluor secondary antibody (Thermo Fisher) and DAPI (Sigma Aldrich) to counterstain the nuclei; after washing 3X with PBS slides were mounted with ProLong™ Gold Antifade mounting solution (Thermo #P36934) and acquired in Z-stack mode with confocal microscope (NIKON Eclipse Ti) using EZ C.1 acquisition software (Nikon, Tokyo, Japan); all the images were processed using FIJI software (ImageJ). The fluorescent intensity profiles of Lamin B1 was performed with NIS elements analysis software 6.0 (Nikon).