Ti microscope

Immunofluorescence analyses were performed using 5 μm sections of FFPE tissues essentially as described.^{32 (link)} Briefly, slides were deparaffinized in xylene and hydrated in a series of descending ethanol. After heat-induced antigen retrieval in TRIS-EDTA (pH = 9) buffer, the samples were permeabilized with 0.5% TritonX-100, blocked with 5% goat serum in phosphate-buffered saline (PBS) and stained with anti-pSTAT3 antibody (Cell Signaling, cat#9131L) using a tyramide-based amplification kit (Perkin Elmer), followed by co-stain with anti-CD8 (ThermoFisher Scientific, cat#MA5-13473) and GranzymeB (Abcam, cat#ab4059) antibodies. Image analysis was performed on 3 × 3 montage images acquired by a Nikon Ti microscope attached to a Yokogawa spinning-disk confocal unit, ×40 Plan Apo objective, and OrcaER camera controlled by the Andor iQ software. pSTAT3 status was determined automatically using imageJ. CD8⁺ T cells were quantified automatically using ImageJ. GZMB⁺CD8⁺ T cells were quantified manually. Average counts of at least three montages per sample were quantified and used to calculate mean primary and metastases number of infiltrating CD8⁺ and percentage GZMB⁺CD8⁺ T cells.

All confocal images were captured using a Nikon Ti microscope equipped with a Yokogawa CSU X-1 spinning disc and an Andor iXon897 EMCCD camera controlled by Andor IQ3 software. Bright field movies of muscle contraction were acquired using the same microscope in wide field mode and images were captured with an Andor Neo sCMOS camera. All live-imaging assays were performed at 37 °C and in a 5% CO₂ controlled and humidified environment.

Cells were plated onto Mat-Tek 35-mm glass-bottom Petri dishes pretreated with poly-d-lysine and grown to ~70% confluency before treatment. Medium was aspirated, and cells were treated with bispecifics or control antibodies in complete growth medium. For soluble ligand uptake experiments, biotinylated soluble ligand was preincubated with streptavidin–647 at 37 °C for 30 min, mixed with bispecific or control antibodies and added to cells. After 24 h of incubation at 37 °C, medium was aspirated, and cells were washed with PBS. Cells were then stained using standard protocols for LysoTracker Deep Red (Invitrogen), DAPI (Cell Signaling Technologies) and primary antibody. Samples were imaged using a Nikon Ti Microscope with a Yokogawa CSU-22 spinning disk confocal and a ×100 objective lens; 405-, 488- and 647-nm lasers were used to image DAPI, primary antibody and LysoTracker, respectively. Images were deconvoluted and processed using NIS-Element (v5.21.03) and Fiji software (v2.1.0) packages.

Confocal images were captured using Nikon Ti microscope equipped with a Yokogawa CSU X-1 spinning disc and an Andor iXon897 EMCCD camera controlled by Andor IQ3 software. Phase-contrast movies of muscle contraction were acquired using the same microscope in Epi-mode and images were captured with an Andor Neo sCMOS camera. All live imaging experiments were performed with 5% CO2 and 37 °C humidified using in-situ microscope setup. Image analysis was performed using FIJI ImageJ V.2.0.0 and Bitplane Imaris 8.4.3 software.

HPAC, PL5, and HPNE cells were plated on glass-bottom imaging plates (MatTek) and incubated for 24 hours at 37°C. Cells were treated with IgG (1 μg/mL) for 30 minutes and washed with media to remove unbound IgG. Bound IgG was detected by the addition of Alexa Fluor 488–conjugated AffiniPure F(ab′)2 fragment goat anti-human IgG, F(ab′)2 fragment specific (Jackson ImmunoResearch, 143225), in Invitrogen Molecular Probes Live Cell Imaging Solution (Thermo Fisher) containing Hoescht blue (2 μg/mL). Cells were imaged on a Nikon Ti Microscope Yokogawa CSU-22 with spinning disk confocal.

Unless otherwise stated, all confocal, epifluorescence and brightfield images were captured using a Nikon Ti microscope equipped with Yokogawa CSU X-1 spinning disc, controlled by Andor IQ2 software. Epifluorescence was imaged using the same microscope in widefield mode. Confocal and widefield images were captured with Andor iXon897 EMCCD and Neo sCMOS cameras, respectively. In all live-imaging assays performed, imaging of myotube contractions, Fluo-3 transients, FM dye, axonal degeneration after H₂O₂ and axonal transport, living samples were maintained in a humidified incubation chamber at 37°C, 5% CO₂. Details of fluorescent labels and excitation and emission parameters are given in supplementary material Table S1 below. Images were analyzed using ImageJ software, except for BTX-HB9::GFP colocalization, which was analyzed using Imaris (Bitplane) Coloc module and 3D rendering and surface modeling, which were performed using Imaris Surpass and Surface functions.