Las x core software

For P7, P10, and P14 animals, three sections equally spaced across the anterior-posterior axis of the DG were selected per animal using Hoechst counterstain with the 10x objective on the epifluorescence microscope. One 63x frame was captured using a confocal microscope (Leica TCS SP8) for each blade (suprapyramidal and infrapyramidal) of the DG of each selected section for a total of six frames. For P163 animals, four sections equally spaced across the anterior-posterior axis of the DG were selected per animal using Hoechst counterstain with the 10x objective on the epifluorescence microscope. The entire DG in one hemisphere of each selected section was imaged on the confocal microscope. Confocal images were visualized using Leica LAS X Core software (http://www.leica-microsystems.com/products/microscope-software/details/product/leica-las-x-ls/). All KOr + GFAP+ cells with radial processes were manually counted in all captured frames and MCM2 co-expression was manually assessed. For P7, P10, and P14 animals, 200 to 300 KOr + GFAP+ radial stem cells were assessed per animal (average: 250 cells/animal). For P163 animals, 150 to 300 KOr + GFAP+ radial stem cells were assessed per animal (average: 220 cells/animal).

Organoids grown on glass bottom chamber coverslips (ibidi) were used for live imaging with Leica SP8 lightning confocal microscope (Leica, Wetzlar, Germany) inside humidified 37°C chambers with 5% CO₂. The imaging protocol was established based on the previously published method.
³⁴ (link) Briefly, before image acquisition, 1 mM lucifer yellow dye (L0144, Sigma Aldrich) was added to the culture media. Images were acquired in 10 random fields with organoids for 20 minutes at 5-minute intervals after dye addition, at ×200 magnification. Image analysis was performed using Leica LAS X core software. Average background baseline fluorescence was measured from images acquired 1 minute after dye addition. For quantification, the background-subtracted mean fluorescence intensities at 20 minutes were measured inside and outside the organoids within a set region of interest. % Dye intensity inside organoids was calculated by dividing fluorescence intensity inside with total intensity inside + outside organoids. Ten random organoid fields were imaged/sampled in 6 experiments.

A cell-based fluorescence (CBF) assay, as previously described by Singh and colleagues [21 (link)], was performed to quantify changes within the glycocalyx using fluorescein isothiocyanate-labelled wheat germ agglutinin (WGA-FITC), which binds to N-acetyl neuraminic acid and N-acetyl glucosamine residues of proteoglycans and glycoproteins present in the glycocalyx.
Lectin-binding (as WGA-FITC binding) was also observed (as previously described [22 (link)]), before and after 1 h treatment with lung tumour CM, with a confocal laser scanning microscope (Leica DMi8 TCS SP8 Confocal, Leica Microsystems, Germany). The images (1024 x 1024 pixels) were acquired with a 10x tube lens and processed using the Leica LAS X Core software.

Proximity ligation assay (PLA) was performed for in situ detection of the distance between ER and mitochondria (distance < 40 nm) using Duolink^® In Situ Orange Starter Kit Goat/Rabbit (Sigma Aldrich, Germany) following the manufacturer’s instructions. Briefly, two primary antibodies raised in different species (goat anti-calnexin as an ER marker and rabbit anti-TOM20 or rabbit anti-VDAC1 as mitochondrial markers, antibody information in Table 1) were used. A pair of oligonucleotide-labeled secondary antibodies (PLA probes) was added to bind to the primary antibodies. Hybridizing connector oligos were then used to join the PLA probes. Upon close proximity, a circular DNA template is formed by ligase activity resulting in rolling-circle amplification. Amplified signal tethered to the PLA probe was generated that allowed signal detection. The labeled oligos that hybridized to the complementary sequences within the amplicon were visualized and quantified by microscopy image analysis. Leica M205 FA fluorescent stereoscope (Leica Microsystems, USA) and LAS-X-Core Software (3.7.4. version, LAS X Life Science, USA) were used. PLA results were quantified by Image J software (version 1.52a, NIH, USA) following user guide (https://imagej.nih.gov/ij/docs/guide/user-guide.pdf; https://www.unige.ch/medecine/bioimaging/files/1914/1208/6000/Quantification.pdf).