Axio observer 7 inverted microscope

Cells were imaged in culture media supplemented with 20 mM HEPES (SH3023701, HyClone) at 37°C, on a Marianas Imaging System (Intelligent Imaging Innovations) consisting of an Axio Observer 7 inverted microscope (Zeiss) attached to a W1 Confocal Spinning Disk (Yokogawa) with Mesa field flattening (Intelligent Imaging Innovations), a Phasor photomanipulation unit (Intelligent Imaging Innovations), a motorized X,Y stage (ASI), and a Prime 95B sCMOS (Photometrics) camera. Illumination was provided by a TTL triggered multifiber laser launch (Intelligent Imaging Innovations) consisting of 405, 488, 561, and 637 lasers, using a 63×1.4 NA Plan-Apochromat objective (Zeiss). Temperature and humidity were maintained using a Bold Line full enclosure incubator (Oko Labs). The microscope was controlled using Slidebook 6 Software (Intelligent Imaging Innovations).

For fusion measurement, samples were prepared in Grace BioLabs CultureWells with coverslip bottom treated with 1% Pluronics F-127 (1 h). Timeseries of fusion events were collected at 49-ms time intervals on a widefield Axio Observer 7 Inverted Microscope (Zeiss) with a ×63/1.4 NA Plan-Apochromat (oil immersion) objective. FITC was excited with a 120-W metal halide lamp (X-cite 120) with 470/40 nm excitation and 525/50 nm emission filters. Images were acquired with a Axiocam 506 mono camera (Zeiss) controlled by the Zen software (Zeiss). ImageJ was used to further format and process images, and MATLAB was used to analyze fusion events as described previously^{69 (link)}.

The specimens were fixed with 4% paraformaldehyde in Phosphate-Buffered Saline (PBS) at 4 °C overnight. The fixed samples were washed with PBS with 0.3% Triton X-100 (PBST), treated with 1.5% normal goat serum in PBST for 1 h, and incubated with rabbit anti-human Frizzled3 polyclonal antibody (CSB-PA882067ESR2HU; Cusabio) at 4 °C overnight. The primary antibody was used at a dilution of 1:50 in PBST containing 0.5% Bovine Serum Albumin (BSA). After a brief rinse with PBST, the samples were reacted at room temperature for 2 h with a secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen), diluted 1:1000 in PBST containing 0.5% BSA. After washing with PBS, the samples were placed with the apical surface downward in a thin-glass-bottomed Petri dish filled with PBS, gently pressed onto the bottom by metal weights, and examined under a Carl Zeiss LSM880 confocal laser-scanning microscope (Carl Zeiss).
The light source, an argon laser, was coupled to an Axio Observer7 inverted microscope (Carl Zeiss). Excitation wavelength was 488 nm. Laser power was reduced to 1.0% by a neutral density filter. An objective was Plan-Apochromat 40× with oil immersion. Emitted fluorescence was detected by a photomultiplier and displayed as a 512 × 512 pixel resolution image using Carl Zeiss ZEN 2 software.

LLP was studied using a fluorescent probe, FOAM-LPO. FOAM-LPO was synthesized as previously described (26 (link)). Cells were cultured on an 8 Well μSlide (ibidi, 80807) and treated with inhibitors and with or without DC661. Cells were incubated with media containing FOAM-LPO (1 M) in an atmosphere of 5% CO₂ and 95% air for 5 minutes at 37°C. The cells were washed twice with media, and then cells were observed under the microscope (Zeiss Axio Observer 7 inverted microscope) to detect fluorescence shifting from 586 to 512 nm in response to LLP.