Lsm 880 airyscan fast confocal microscope

Sample preparation for Airyscan confocal immunofluorescence microscopy. Mice 6 weeks of age were transcardially perfused with 80 mM PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl₂, 4% paraformaldehyde). Eyes were enucleated, and after the cornea was removed, they were immersion fixed overnight at 4 ^oC. After removal of lens, the eyecups were flash-frozen in optimal cutting temperature using liquid nitrogen. 8 μm cryosections were collected and stained for rhodamine wheat germ agglutinin (Vector RL-1022) and phalloidin conjugated to Atto647N (Sigma 65906) or cofilin (CST 5175) and phalloidin conjugated to Atto647N (Sigma 65906). The sections were then mounted in Prolong Glass (Invitrogen P36980). Sections were imaged on a Zeiss LSM 880 Airyscan Fast Confocal Microscope using a 63x objective. Z-stacks were first processed in Zeiss ZenBlue software for Airyscan processing, then colour processing and analysis were performed in Fiji/ImageJ. Actin puncta quantification was performed by taking a 0.35 μm ROI around each actin puncta that was at the base of an outer segment, slice by slice, on each z-stack. Then the averaged relative integrated density of three background ROI’s were subtracted from each relative integrated density measurements from the actin ROIs. These measurements were then plotted in Prism software, where Students t-test statistical analysis was performed.

CT26WT cells were seeded on cover slips (No. 1014/10. Assistent, Sondheim, Germany) and treated with PDT or VEGF₁₂₁/rGel-PCI as indicated. Cells were fixed in 4% paraformaldehyde (PFA) and stained with Phalloidin-iFluor 594 Reagent from Abcam (#ab176757 1:1000 in PBS) before they were stained with primary antibodies αHMGB1 (#ab18256, Abcam 1:1000) or αHSP90 (#4877 CST 1:100) over night at 4°C, incubated with secondary Goat αrabbit Alexa 488 antibody (#A11034, Life Technologies1:600 in 1%BSA), incubated 2 min with 0.6 µg/ml Hoechst 3325 (Sigma Aldrich), and mounted using ProLong Glass Antifade mountant (Thermo Fisher). The cells were subjected to microscopy using a LSM 880 Airyscan FAST confocal microscope equipped with an Airyscan detector and FAST options, Ar-laser multiline (405/458/488/514/561 and 633 nm and 20x NA 0.8 DIC II (Plan-Apochromat) and 63x NA 1.4 oil DIC III (Plan-Apochromat) objectives (Carl Zeiss AG, Oberkochen, Germany). The Zen blue software (Carl Zeiss AG) was used for image acquisition and processing. For more details, see Supplementary Material.

Adherent cells were imaged with AxioObserver Z spinning disk microscope equipped with Evolve EM-CCD camera (Photometrics), LSM880 Airyscan Fast confocal microscope, and LSM780 confocal microscope, (all from Carl Zeiss, Germany), with 63x/1.4 oil objectives using standard filter combinations for green, red and far red fluorescence to acquire SYBR Gold and SYBR Green signals (green), Mitotracker CMXRos Red and PI (red) and Mitotracker Deep Red signal (far red), except specially described cases. Signals in different color channels were acquired sequentially.