Evos m7000 microscope

The click-iT EdU cell proliferation kit (Thermo Fisher) was applied according to manufacturer’s instructions for the detection of proliferating cell portions. In brief, 1 × 10⁵ cells/well were seeded in 24-well plates and cultured overnight before treatment. After refreshing media on the next day, cells were labelled with 100 µM ethynyl deoxyuridine (EdU) and subsequently exposed to mebendazole (Selleckchem) or dimethyl sulfoxide (DMSO) for 24 h at 37 °C. After 3.7% paraformaldehyde/PBS fixation and 0.5% Triton-X permeabilization, cells were stained with Alexa Fluor 555 azide for 30 min. Hoechst 33342 was used for staining of nuclei. Images were captured with the 10× objective of the EVOS M7000 microscope (Invitrogen) and EdU-positive nuclei counted in relation to the total number of Hoechst 33342-positive nuclei using the EVOS software.

Briefly, 2 × 10⁵ cells were seeded in µ-slide 8-well chambered coverslips (Ibidi, Gräfelfing, Germany) and incubated in culturing medium containing either DMSO or mebendazole for 24 h. In addition, 5 µm cryosections of tumor samples of the animal studies that have been air-dried for 10 min were used. Cells or tissue sections were fixed in 4% paraformaldehyde/PBS for 15 min, permeabilized with 0.5% Triton X-100 for 1 h and then blocked in PBST containing 3% BSA and 0.1% glycine for 10 min. Slides were then incubated overnight at 4 °C with mouse anti-α-tubulin (clone DM1A) (Sigma-Aldrich), Ki67 (Abcam, Cambridge, UK) and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA) antibodies in 5% BSA/PBS at dilutions of 1:100, 1:250, and 1:100, respectively. The next day, following serial washing steps, slides were incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen) in a dilution of 1:250 in 5% BSA and 0.1% Triton X-100. Slides were counterstained with 1 µg/mL Hoechst 33342 (Invitrogen) at room temperature in the dark for 1 h. Images of tubulin staining were captured with the 40× objective of the Axiovert 200M microscope equipped with an AxioCam MRm (Zeiss, Jena, Germany) and abnormal spindles were counted by ImageJ. Ki67 and caspase-3 stainings were captured with the EVOS M7000 microscope (Invitrogen).

For H&E staining, mouse ears and tails were fixed in 4.5% formaldehyde overnight and embedded in paraffin. Rewaxed and rehydrated tissue sections (2 μm) were stained with Gill III Hematoxylin for 2.5 minutes, washed, and stained with 0.5% eosin Y for 1 minute afterward. Slides were placed in 95% ethanol 2 times and transferred into 100% ethanol for 2 minutes. Slides were incubated with xylene I/II for 2 minutes, dried, and covered with mounting medium (Consul Mount, Thermo Fisher Scientific). Pictures were taken with an Invitrogen EVOS M7000 microscope.
For immunofluorescence histology, cryosections of skin were fixed and stained with CryoFixation and CryoStainer of xZell, as recommended by the manufacturer (xZell, Singapore). To detect Treg populations, sections were incubated with PE Foxp3 antibody. DAPI was used to stain nuclei. Immunofluorescence imaging was performed with Leica Thunder 3D Tissue Imager. Pictures were analyzed with ImageJ (NIH).

ADAR1 knockout HEK293T cells (5 × 10⁶ cells/10 cm² plate) were co-transfected with huMycADAR2, huMycADAR1-p110 or huMycADAR1-p150 (2μg), and mCherry_2PA_eGFP(W58X)_pcDNA3.1(–) (10μg) plasmids to express human ADAR2, ADAR1-p110 or ADAR1-p150, and eGFP containing the early termination mutation W58X. After 24 h, cells were detached with 0.05% trypsin-EDTA (Gibco, Catalog number: 25300-054) and 5000 cells/well were reverse transfected with oligonucleotides in 384-well plates. Both plasmid and oligonucleotide transfections were performed with Lipofectamine 3000 (ThermoFisher, Catalog number: L3000-015) diluted in OptiMEM (Gibco, Catalog number: 11058–021) following the manufacturer's instructions. After 48 h, eGFP fluorescence was imaged using an EVOS M7000 microscope (Invitrogen) and quantified with a BioTek Cytation5 imaging reader with excitation/emission wavelengths at 488 nm/510 nm, respectively, before cells were lysed and mRNA was isolated using Dynabeads™ Oligo(dT)₂₅ (ThermoFisher, Catalog number: 61005). Isolated mRNA was cleaned with ezDNase to avoid genomic and plasmid DNA contamination and then reverse transcribed to cDNA with SuperScript IV Vilo (ThermoFisher, Catalog number: 1176500) before proceeding to next generation sequencing (NGS).

The phagocytosis assay was performed using pHrodo green E. coli bioparticles (Invitrogen Cat# P35366) according to the manufacturer’s instructions. Five images per well were then collected every 30 minutes for 24 hours on an EVOS M7000 microscope (Invitrogen, ThermoFisher Scientific). The cells were maintained for this duration using an on-stage incubator kept at 37°C and 5% CO₂. Analysis of the collected images was performed using EVOS Celleste software (version 5.0), which counted the cells and then measured the integrated optical density of fluorescence within each well over the 24-hour time course. Analysis was conducted on the average integrated optical density across triplicate wells. Results were consistent across duplicate experimental replicates.

1 × 10⁴ cells/well were seeded in 6-well plates and incubated overnight. The next day, cells were changed into culturing media containing either DMSO or 0.5 µM mebendazole for 5 days. Upon formation of colonies, cells were fixed with ice-cold methanol for 15 min and stained with 0.5% crystal violet (Sigma-Aldrich) in 20% methanol for 1 h. Pictures were taken with using the 4× objective of the EVOS M7000 microscope (Invitrogen) and colonies irrespective of their size counted with ImageJ “https://imagej.nih.gov/ij/” (accessed on 25 April 2021).

We used the same method as described in our recent report (Zhang et al, 2018 (link)). Briefly, artery paraffin sections were de-paraffinized and subjected to antigen retrieval. Following blocking, a primary antibody was added and incubated overnight. The sections were rinsed and incubated in a fluorescence-labeled secondary antibody for an hour. Detection of immunofluorescence was performed under an EVOS M7000 microscope (Thermo Fisher Scientific). For quantification, 3–5 immunostained sections from each animal were used. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) for counting cell numbers. Fluorescence intensity in each image field was quantified by using ImageJ software (National Institutes of Health) and normalized to cell number. The values from all sections of each animal were pooled to generate an average value. The averaged values from all the animals in each treatment group were averaged again to produce mean ± SEM.