Imagej

Zeiss LSM 880 confocal system equipped with Airyscan detector, using 488, 561, and 633-nm laser lines for green, red, and far-red fluorescent probes, respectively. Images were taken through a ×63 oil objective (numerical aperture [NA] 1.40). Images were captured using Carl Zeiss ZEN 2.3 software package (Carl Zeiss Microscopy GmbH 1997–2015) and were processed using ImageJ (Wayne Rasband, National Institutes of Health, USA).
FRAP experiments were performed on a Zeiss LSM780 NLO system using a 63x oil immersion objective (PlanApo, 1.4 N.A.). DPSS laser at 561 nm was used for the excitation of mRFP/mCherry. Circular regions 5 µm in diameter were photobleached by pulsed Ti:Sapphire laser (Coherent Chameleon Vision II) at 720 nm to achieve rapid, extensive bleaching. Images were acquired at 1 frame/s using Carl Zeiss ZEN 2.3 software package (Carl Zeiss Microscopy GmbH 1997–2015) and were processed using ImageJ (Wayne Rasband, National Institutes of Health, USA).

Fluorescent images were obtained using an LSM710 Confocal Microscope (Zeiss) with 25× (for adult pituitary) and 40× (for juvenile pituitary) objectives. Lasers with wavelength of 405 (DAPI), 555 (TAMRA; Alexa-555), 633 (Cy5) and 488 (FITC; Alexa-488) nm were used. Channels were acquired sequentially to prevent cross-signaling of fluorophores. Due to the size of the adult pituitaries, the image acquisition was done from the dorsal and ventral sides of the pituitary with some overlaps in the middle. In conjunction with the microscope, ZEN software (v2009, Zeiss) was used to process the images, and ImageJ (1.52p; http://rsbweb.nih.gov/ij/) was used for processing z-projections from confocal image stacks. The dorsal and ventral stacks of adult pituitaries were aligned using HWada (ImageJ/635/" xlink:type="simple">https://signaling.riken.jp/en/en-tools/ImageJ/635/) and StackReg plugins (http://bigwww.epfl.ch/thevenaz/stackreg/), before presenting them in orthogonal views.

Passage 4 un-pooled HCAECs (Sigma-Aldrich, United Kingdom) were seeded in 24-well plates with MEGM (Sigma-Aldrich, United Kingdom) either with or without 5% pooled pre- or post-exercise intervention serum and incubated at 37°C in 5% CO₂ for 24-h, as outlined above. HCAECs were mechanically scraped using a 200 µL pipette to create a vertical wound in the confluent monolayer, washed with 200 µL PBS to remove debris, and 500 µL fresh MEGM (Sigma-Aldrich, United Kingdom) added to each well. HCAECs were then exposed to relevant concentration of chemotherapy drugs as described before.
Cells were imaged using Primovert Axiocam ERc5s microscope (ZEISS, Germany) using ×4 magnification at 0, 3-, 4-, 6-, 12-, 24- and 48-h and analysed using ImageJ (1. x, Java, United States). Rate of wound closure was determined by the area of the wound plotted as a function of time. Area under the curve (AUC) was calculated using known equations (Jonkman et al., 2014 (link)).

Imaging of SCMS, HEK293 cells, and hippocampal neurons was done on a Zeiss LSM 510 confocal laser‐scanning microscope, equipped with an oil immersion objective, numerical aperture 1.4, 63× (Zeiss). For excitation of YFP, a 488 nm argon‐krypton laser was used, with detection using a 505–550 nm bandpass emission filter. Red dyes were excited using a 543 nm helium‐neon laser, and emission was detected using a 560–615 nm emission filter. Far red fluorophores were excited using a 633 nm laser light from a helium‐neon (HeNe) laser source, and emitted fluorescent light was filtered using a long pass filter.
Images were acquired at 1,024 × 1,024 pixels, 8‐bit pr. pixel, with 4‐, 8‐, or 16‐line averaging scans. Images were treated using ImageJ or Zen (Zeiss software).

Immunofluorescence images were quantified by determining the MFI of lens capsule–associated tissue viewed in 3 randomly chosen confocal images from biologically independent samples using ImageJ (v1.52P, NIH) (73 (link)). The average number of lens capsule–associated nuclei/section was analyzed by ImageJ using 6 randomly chosen immunofluorescence images from each PCS time point from at least 3 biologically independent samples as described (73 (link), 74 (link)).
The diameter of adult lenses was determined by dissecting both lenses from 3 WT and 3 β₈ITG-cKO mice and photographing them in brightfield using a Zeiss STEMI SV 11 dissecting microscope. The diameter of each lens was measured in 2 perpendicular axes using ImageJ, then averaged for statistical analysis.
All statistics were assessed using either 2-tailed Student’s t test (corrected for multiple comparisons using the Holm-Šídák method) or 1-way/2-way ANOVA with Tukey’s post hoc test using GraphPad Prism 8.3.0/9.2.0. Data are presented as mean ± SEM, and differences were considered significant at P ≤ 0.05.

Examination of gene expression by whole-mount in situ hybridization was performed essentially as previously described [33 (link)–37 (link)]. Prior to mRNA in situ hybridization analyses, embryos were fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) overnight at 4°C or 4–5 hours at RT with gentle agitation on a rotating platform. Embryos were permeabilized in 10 μg/ml proteinase K for 10 seconds (10–12 hpf embryos), 30 seconds (14–17 hpf embryos), 3 minutes (24–32 hpf embryos), or 1 hour (3–4 days post fertilization embryos) at RT.
Following in situ hybridization, embryos were manually deyolked, and cleared in 30%, 50%, and 70% glycerol/PBS. Mounted in situ hybridized embryos and live Tg(gata1:DsRed)^sd2Tg embryos were photographed using a Zeiss AxioImager Z1 compound microscope with an Axiocam HR digital camera. Mounted Tg(kdrl:GFP)^la116Tg embryos were photographed using a Zeiss LSM510 confocal microscope. Whole embryos were photographed using an Olympus stereoscope with a QImaging micropublisher camera. Images were assembled in ImageJ or Zen (Zeiss), and figures were assembled in Photoshop (Adobe).