Eclipse ti u epi fluorescence microscope

Images of large field were taken by Nikon ECLIPSE Ti-U epi-fluorescence microscope by 10×objectives, and analysed by NIS-Elements F 3.0 software (Nikon, Japan). Besides, images of high resolution were acquired by using OLYMPUS FLUOVIEW FV1000 Confocal Microscope at three channels (FITC, TRITC, DAPI), and analyzed by FV10-ASW 4.0 Viewer software (Olympus, Japan).

Cell-in-cell time-lapse assay was performed as previously described^{2 (link)}. About 3 × 10⁵ cells were suspended in 0.5% agarose-coated plates for 6 h, and then cell suspensions were transferred and grown on cover-glass dishes. Images of cells were captured for DIC and fluorescence channels (excitation of 440 and 550 nm and emission of 610 nm) every 10 min with ×20 objective lenses at 37 °C and 5% CO₂ for 24 h by the Nikon ECLIPSE Ti-U epi-fluorescence microscope and analyzed by NIS-Elements F 3.0 software (Nikon, Japan). The timing of cell death was judged morphologically by the appearance of a broken cell membrane, or cessation of cell movement, or both.

Cells overexpressing fluorescent protein keima were incubated in a series of buffers with pH values ranging from 4.12 to 7.97, then the fluorescent signal of keima was measured under the conditions of excitation of 440 and 550 nm and emission of 610 nm using Nikon ECLIPSE Ti-U epi-fluorescence microscope. Then the fluorescent intensity of keima was measured by NIS-Elements F 3.0 software^{37 (link)}. The pH titration curves and fitting equation were obtained by the negative correlation between the pH value and the fluorescent intensity ratio of 550 nm/440 nm of keima protein.

Immunostaining was performed as previously described^{38 (link)}. Briefly, cells were fixed in 4% paraformaldehyde for 10 min at room temperature, then permeabilized with 0.2% Triton X-100/PBS for 3 min and washed with PBS followed by blocking with 5% BSA at room temperature for 1 h. Fixed samples were incubated with primary antibody at 4 °C overnight and washed with PBS before incubated with fluorophore-labeled secondary antibodies at room temperature for 1 h. Cells mounted with mounting medium with DAPI (ZSGB-BIO, #ZLI-9557) and imaged by Nikon ECLIPSE Ti-U epi-fluorescence microscope.

Formaldehyde-fixed paraffin-embedded sections were steamed in IHC-Tek Epitope Retrieval Solution (IHCWorld) for 40 minutes, then blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% donkey serum. Sections were incubated overnight at 4°C with a goat anti-mouse NPNT polyclonal (R&D, catalog number AF4298) or IgG isotype control (Jackson Immunoresearch, catalog number 005-000-003) at 2 μg/ml in TBST containing 1% BSA. Staining was visualized using Alexa Fluor555-conjugated donkey anti-goat (Invitrogen) antibody at 4 μg/ml and images were captured at an equivalent laser intensity with a Nikon Eclipse Ti-U epifluorescence microscope.

Immunostaining was performed as previously described^{59 (link)} with the following steps: cells were fixed with 4% paraformaldehyde for 10 min at room temperature, then permeabilized with 0.2% Triton X-100/PBS for 3 min, and washed with PBS followed by blocking with 5% BSA for 1 h. The fixed samples were incubated with primary antibodies dissolved in 5% BSA at 4 °C overnight, washed with PBS three times, and incubated with secondary antibodies for 1 h at room temperature. After washing with PBS three times, samples were mounted with mounting medium (ZSGB-BIO, #ZLI-9557) and imaged by Nikon ECLIPSE Ti-U epi-fluorescence microscope. Immunoblotting was performed as previously described^{60 (link)} with the following steps: total proteins were extracted and quantified with Pierce BCA Protein Assay KIT (Thermo, #23227), then subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The transferred samples were blocked with 5% bovine serum albumin (BSA) and probed with primary antibodies and species matched secondary antibodies, then visualized by chemiluminescence SuperSignal KIT (Thermo, #34095) according to the manufacturer’s instructions.