Emulsion samples were collected on a glass microscope slide and heated using a precision Peltier heating and cooling stage (Linkam LTS120) equipped with a Linkam PE95 digital temperature control unit. The spatial distribution of Alexa Fluor 488-labeled (25% molar fraction N-terminal labeled) ELP/ELP-RBD and Alexa Fluor 594-labeled (5’ conjugated) ssRNA1 was characterized via fluorescence microscopy using an upright Zeiss Axio Imager D2 microscope with a 20× objective and the appropriate filter set (ex 470/40, em 525/50). Similarly, fluorescence intensity of superfolder GFP over time was characterized via fluorescence microscopy using an upright Zeiss Axio Imager D2 microscope with a 20× objective and the appropriate filter set (ex 470/40, em 525/50). Coacervate formation was monitored via bright-field microscopy using an upright Zeiss Axio Imager D2 microscope with a 20× objective. Fluorescence intensity within droplets was characterized using MATLAB.
Axio imager d2 microscope
Manufactured by Zeiss
Sourced in Germany
The Axio Imager D2 is a versatile upright microscope designed for a wide range of applications. It features a modular design and offers a variety of observation techniques, including brightfield, darkfield, phase contrast, and differential interference contrast. The microscope is equipped with high-resolution optics and a robust mechanical structure to ensure stable and precise imaging.
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Lab products found in correlation
Lts120, by Linkam (3 mentions)
Plan apo 1.4na, by Zeiss (3 mentions)
Fitc conjugated goat anti rabbit secondary antibody, by MultiSciences Biotech (2 mentions)
Rm2125rt microtome, by Leica (2 mentions)
Axiocam hrc, by Zeiss (2 mentions)
Evos xl cell imaging system, by Thermo Fisher Scientific (2 mentions)
Colcemid, by Roche (2 mentions)
Telomere repeat specific peptide nucleic acid probes, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Nucleopore, by Cytiva (1 mentions)
Collibri led module illumination system, by Zeiss (1 mentions)
β 3 tubulin antibody, by Abcam (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Goat serum, by Merck Group (1 mentions)
Axio cam 305 camera, by Zeiss (1 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
Plan neofluar, by Zeiss (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Nuclear fast red, by Merck Group (1 mentions)
36 protocols using axio imager d2 microscope
1
Visualizing ELP-RBD and ssRNA1 Coacervation
2
Retinal Cell Quantification in rd10 Mice
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Eyes were harvested from euthanized rd10-non, rd10-SA4503, rd10-PRE084, and rd10-(+)-PTZ mice (age P42). They were either processed for embedding in JB-4 methacrylate or flash frozen for cryosectioning as described.40 Sections were viewed using a Zeiss Axio Imager D2 microscope (Carl Zeiss) equipped with a high-resolution camera and processed using Zeiss Zen23pro software. Given extensive PRC loss in the rd10 mouse, we measured ONL thickness and also counted the total number of PRC nuclei along the full length of retina (from the temporal to nasal ora serrata) in three retinal sections per mouse. Nuclei counts were averaged per mouse and per treatment group. To evaluate whether treatment with the different Sig1R ligands was associated with decreased oxidation in retina, cryosections were incubated with hydroethidine as reported13 (link) (dihydroethidium, Molecular Probes/ThermoFisher). Retinal cryosections were viewed by epifluorescence using the Zeiss Axio Imager D2 microscope. To evaluate whether cone PRCs were preserved, retinal cryosections were subjected to FITC-labeled peanut agglutinin (PNA) (Millipore/Sigma), which selectively binds cone inner/outer segments. Earlier reports showed increased PNA-labeling in (+)-PTZ-treated rd10 mice compared with nontreated mice.13 (link)
3
Characterization of Protein Spatial Distributions
4
Epifluorescence Microscopy for Bacterial Abundance
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Samples of 50 ml were fixed with buffered, sterile‐filtered paraformaldehyde (Penta, Prague, Czechia) to a final concentration of 1%, and 0.5 ml was filtered onto white polycarbonate filters (pore size 0.2 μm, Nucleopore, Whatman, Maidstone, UK). Cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) at concentration of 1 mg L−1 (Coleman, 1980 ). Total and AAP bacterial abundances were determined using an epifluorescence Zeiss Axio Imager.D2 microscope equipped with Collibri LED module illumination system (Carl Zeiss, Jena, Germany). Ten microphotographs were taken for every sample under 325–370 nm excitation and 420–470 nm emission wavelengths for DAPI fluorescence (total bacteria), 450–490 nm excitation and 600–660 nm emission wavelengths for autofluorescence from Chl‐a (algae and cyanobacteria), and combined 325–370 nm, 450–490 nm, 545–565 nm and 615–635 nm excitation and 645–850 emission wavelengths for autofluorescence from BChl‐a (AAP bacteria). As some part of Chl‐a autofluorescence is also visible in the infrared spectrum, only the IR‐positive cells that did not show any autofluorescence from Chl‐a were counted as AAP bacteria (Cottrell et al., 2006 (link)).
5
Immunofluorescence Analysis of Retinal Ganglion Cells
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After 2, 4, and 7 days, RGCs on coverslips were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) at room temperature for 15 minutes, followed by washing three times with PBS. Cells were then membrane permeabilized with 0.3% triton X-100 in PBS at room temperature for 10 minutes. Next, cells were blocked with 0.1% triton X-100 in PBS with 10% goat serum (Sigma) at room temperature for 1 hour, then incubated in βIII-tubulin antibody (1:500, Abcam, Cambridge, MA) at 4 °C overnight. Next, cells were incubated in secondary antibody (Alexa Fluor 555-labeled goat anti rabbit 1:1000; Invitrogen) at room temperature for 2 hours. After washing three times with 0.1% triton X-100 in PBS, coverslips were mounted with Fluoroshield with DAPI (Sigma). Cells were observed for immunofluorescence using a Zeiss Axio Imager D2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with Zeiss Zen23pro software and a high-resolution camera. RGCs neurites were traced and evaluated using the Simple Neurite Tracer plugin within ImageJ software.45 (link)
6
Neuroprotective Effects of Sigma-1 Receptor Ligands
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The capacity of SA4503 and PRE084 to mitigate oxidative stress was compared with that of (+)-PTZ. The 661W cells were seeded on coverslips and exposed to tBHP (55 µM) for 2 hours in the presence or absence of SA4503 (3, 50, and 100 µM), PRE084 (3, 50, and 100 µM) or (+)-PTZ (3 and 50 µM). Control cells received no tBHP nor treatment with Sig1R ligands. To detect intracellular reactive oxygen species (ROS), cells were incubated with 5 µM CellROX Green Reagent (ThermoFisher). CellROX Green Reagent is a cell-permeant dye that fluoresces weakly in a reduced state but brightly upon oxidation by ROS with an absorption and emission maxima of approximately 485 and 520 nm, respectively. It is used in living cells to detect hydroxyl, peroxyl, peroxynitrite, and hydroxyl radicals. DAPI was used to detect cell nuclei. Immunofluorescent detection was performed using a Zeiss Axio Imager D2 microscope (Carl Zeiss, Göttingen, Germany) and Zeiss Axiocam 305 camera equipped with ZenPro software. Fluorescence intensity was quantified using National Institutes of Health Image J 1.48v software (National Institutes of Health, Bethesda, MD). For this analysis, three independent experiments were performed with three repetitions per assay.
7
Quantitative Optic Nerve Imaging
8
Retinal Morphometric Analysis via Cryosections
9
WISH Staining and Paraffin Sectioning
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After WISH staining, 5-day-old embryos were prepared for embedding in paraffin, transferred to paraffin, oriented, sectioned in ∼5 µm transversal sections, and then counterstained using Nuclear Fast Red (Sigma-Aldrich, N3020). Representative pictures were taken using an upright Zeiss Axio Imager D2 microscope (Carl Zeiss) with a Plan-Apochromat 20×/0.75 NA or a dry Plan-Neofluar 40×/0.75 NA objective lens. All images were acquired using an AxioCam HRc full color CCD camera with a 1388×1040 pixel imaging field. Zeiss ZEN blue pro 2011 software package was used for analysis.
10
Nrf2 Nuclear Translocation Assay in A549 Cells
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For Nrf2 nuclear translocation assay, A549 cells (1 ⋅ 10 4 cells/well) seeded onto glass slides (9 mm, diameter) were incubated in 5% CO 2 at 37°C overnight. Then, A549 cells were infected with H9N2 viruses and treated with or without arctiin. After 24 h, the cells were washed three times with PBS, xed with methanol for 20 min, and permeabilized with 0.5% Triton X-100 for 15 min. Then, they were blocked with 5% BSA for 30 min at room temperature and stained with rabbit polyclonal anti-Nrf2 antibody (1:200) overnight at 4℃. Next day, the cells were incubated with FITC-conjugated goat anti-rabbit secondary antibody (1:200; MultiSciences, Hangzhou, China) for 1 h at room temperature, and cellular nuclei were stained with DAPI. The glass slides were sealed with glycerin, and imaging was performed with ZEISS Axio Imager.D2 microscope (ZEISS, Hamburg, Germany).
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