Application suite x las x

Tissue samples were collected and fixed in 10% neutral buffered formalin and then processed by the Histopathology Core Facility in the University of Sydney. H&E staining, Picro-Sirius red staining and immunohistochemistry were completed as described previously [22 (link),28 (link)]. Images were captured with a Leica DM6000B microscope (Wetzlar, Germany) and analysed with automation provided by Leica Application Suite X (LAS X) version 3.5.5.19976. Stain area thresholding was varied according to the isotype negative control immunostain. The immunostained area of CD8 or CD4 was measured using a threshold value and divided by total tissue area to calculate the proportion of positive staining on each section [22 (link),30 (link),31 (link)]. Histological quantification of lesions was performed as described [22 (link)].

Cells were mounted on a 2% agarose pad containing resuspension medium using a gene frame (Bio-Rad, Hercules, CA, USA). Cells were concentrated by centrifugation (3,300 g for 30 seconds) prior to mounting and visualization. This step had no impact on the localization of the fusion proteins. Fluorescence microscopy was performed using a Leica (Buffalo Grove, IL, USA) DMi8 wide-field inverted microscope equipped with an HC PL APO 100×DIC objective (NA = 1.40) and an iXon Ultra 888 EMCCD Camera from Andor Technology (Belfast, Northern Ireland). Membranes were stained with TMA-DPH at a final concentration of 100 μM. Excitation light intensity was set to 50%, and exposure times were 300 ms for TMA-DPH (λ_ex = 395/25 nm; λ_em = 460/50 nm); 500 ms for m(E)GFP (λ_ex = 470/40; λ_em = 500 to 550); and 1 second for mYFP (λ_ex = 510/25; λ_em>530) respectively. Images were acquired with Leica Application Suite X (LAS X), and analysis and processing were performed using the ImageJ software [97 (link)].

Cells seeded on coverslips for more than 24 h were fixed in 4% formaldehyde for 30 min at room temperature, then permeabilizated with 0.1% Triton X-100 for 30 min. After that, cells were treated with the primary antibody overnight at 4°C, followed by incubation with species-specific secondary antibody for two hours at room temperature. The coverslips were mounted using an antifade mounting solution containing 4,6-diamidino- 2-phenylindole (DAPI; P36935, Invitrogen) after three-time washing with phosphate-buffered saline (PBS). Actin was stained with Myo10 (Rabbit, HPA024223, Sigma-Aldrich) and Rhodamine Phalloidin (R415, Invitrogen) following the manufacturer's instructions. Confocal images were captured using ZEISS LSM 880 confocal microscope with Airyscan (Carl Zeiss, Germany) or Leica TCS SP8 confocal microscope (Leica, Switzerland), and fluorescence quantification was measured as described previously 9 (link). Co-localization analysis was performed via Leica Application Suite X (LAS X).

Three representative areas were imaged using a bright field microscope (Leica DMi8) with Leica Application Suite X (LAS X). For nuclear staining (Ki67 and phospho-STAT3), images were converted to 8-bit images and threshold adjusted. Then positive nuclear staining was counted and analyzed with “analyze particles, Size (pixel^2: 10-Infinity)”. MECA-32 stained vessels and lung nodules from tail vein injection were manually counted. Overlapping regions (MAFF, HIF-1, BACH1, IL11, pimonidazole) were calculated after drawing the region of interest and compare the percentage of positively stained areas, which were coexisted in both tissue sections. For spontaneously metastasized lung nodules, whole tissue sections were imaged using NanoZoomer 2.0-RS (Hamamatsu). Then using ImageJ, images were converted to 8-bit images and contrast was enhanced to saturated pixels of 0.35%. After threshold was adjusted, images were mask converted to generate to black and white images. From converted images, lung nodules were counted using “analyze particles, Size (pixel^2: 5-infinitiy)”.

Gross images were taken with a Sony Macro Lens and camera. Figure 1a was created with https://www.biorender.com/. IHC images were captured using Leica STELLARIS 5 confocal system (using the 10 × objective) with Leica Application Suite X (LAS X) at the CHP Rangos imaging core facility. Brightfield imaging was obtained using the Thermo Fisher EVOS M7000 system (using the 10 × and 20 × objectives) with EVOS M7000 Software revision 2.0.2094.0. Whole H&E slides were scanned using the Leica Aperio AT2 system (using the 40 × objective) and subsequently evaluated using Aperio ImageScope 12.4.3. Images were optimized using Adobe Photoshop (version 23.3.1). Figures were compiled using Adobe Illustrator (version 26.2.1).