Axio imager upright microscope

Cells plated on glass coverslips were fixed with 3.7% formaldehyde and
then permeabilized with 0.5% Triton X-100 for 10 min. For
calnexin staining, cells were permeabilized with ice-cold methanol for
10 min. Cells were blocked and washed in 1% BSA,
0.1% Tween-20 in 1 × phosphate buffered saline (PBS)
(PBTA). Cells were incubated with primary antibodies for 1 h followed
by three washes in PBTA. Coverslips were then incubated with Alexa
Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for
30 min followed by three washes in PBTA and a wash in H₂O.
MitoTracker was added for 30 min prior to fixation. Prolong Gold
Antifade reagent with DAPI (Thermo Fisher Scientific) was used to mount slips on
glass slides and images were acquired on either a Leica DM55000 B upright
microscope, a Leica TCS SP5 confocal microscope, a Zeiss Axioimager upright
microscope or a Zeiss LSM510 confocal microscope.

Bacterial cultures in logarithmic or stationary phase were incubated in medium for 1 h with 10 μM HS-198 and wheat germ agglutinin (WGA) conjugated with Alexa 488 at 10 μg per ml (Biotium catalog number 29022), and cells were then washed with fresh medium and mounted with Prolong Gold with 4′,6-diamidino-2-phenylindole (DAPI). Samples were imaged on a Zeiss Axio Imager upright microscope, using 63× oil objective, and visible light for differential inference contrast (DIC) imaging and excitation at 405, 488, or 633 nm for detection of DAPI, fluorescein isothiocyanate (FITC), or HS-198, respectively.

To observe root cell morphology of Arabidopsis under salt stress, four-day-old seedlings were transferred to 100 mM NaCl medium for 7 days. Roots were then excised and root tips were observed with differential interference contrast (DIC) under bright field using Axio Imager upright microscope (Zeiss). For the detection of subcellular localization, the roots of eight-day-old transgenic Arabidopsis, or the Nicotiana Benthamiana leaves that were transformed with plasmids for 2 days, were observed using confocal laser scanning microscopy (TCS SP8, Leica).

iPSC‐derived RPE cells grown on 24‐well PET hanging cell culture inserts (Merck) (pore size 0.4 μm) were fixed with 4% paraformaldehyde (PFA) (Sigma, 47608) for 20 min at room temperature. Before blocking, the pigmentation of RPE cells was removed using Melanin Bleach Kit (Polysciences), followed by 3 washes with PBS. RPE cells were blocked in PBS supplemented with 10% donkey serum and 0.3% Triton‐X100 (Sigma, T8787) to permeabilised the cells, for 1 h at room temperature. For p62 and LC3, methanol fixation was used instead and no bleaching prior to immunostaining was carried out. The RPE cells were incubated with primary antibodies overnight at 4°C (Table S1). Following three washes with PBS, RPE cells were incubated with secondary antibodies (Table S1) diluted in solution (PBS, 1% donkey serum, and 0.1% Triton X‐100) and stored overnight at 4°C. Nuclei were stained with Hoechst (Life Technologies, UK). Then, RPE cells were mounted with Vectashield and sealed with a coverslip. RPE cells were imaged using the Axio Imager upright microscope with Apotome structured illumination fluorescence using 20× objective, 40× and 63× oil objectives (Zeiss, Germany). Images were presented as a maximum intensity projection and adjusted for brightness and contrast in Adobe Photoshop (Adobe Systems).

Tissue sections or SiHa and A549 cells grown on coverslips were permeabilized in 0.1% Triton and incubated with mouse anti-γH2AX antibody and rabbit antibodies against H3p, β-tubulin, and Chk2^T68 (listed in Supplementary Table S2). Secondary labeling employed anti-rabbit and anti-mouse antibodies conjugated to Alexa Fluor® 488, 568 or 647, and nuclei were counterstained with DAPI. Images were observed using an Olympus FV1000 upright confocal microscope or Zeiss AxioImager upright microscope and captured at 405 nm, 488 nm, 568 nm and 647 nm using 40x objective lens, before processing with FV10-ASW 3.0 Viewer or AxioVision software.

Amyloid plaques, immunostained with 4G8, were viewed with an Axio Imager upright microscope (Carl Zeiss Microimaging GmbH, Göettingen, Germany) equipped with an AxioCam MRc color digital camera. Stereology-based quantification of amyloid burden was carried out using Stereo Investigator 9 software (MBF Bioscience, Chicago, IL). The entire cerebral cortex and hippocampus were separately sampled with the counting frame size 250 μm × 250 μm for cortex and 100 μm × 100 μm for hippocampus. The sum of the area of all amyloid plaques was divided by the total area of cerebral cortex or hippocampus to obtain the amyloid burden. Experimenters performing quantification of amyloid burden were blind to age and genotype of mice.