Cells were plated in chamber slides and incubated at 37°C overnight for attachment. The cells in the chamber slides were washed 3 times in PBS and fixed in cold acetone for 15 min at room temperature and then washed twice with PBS for 5 min. After washing, the cells were blocked with 0.05% BSA in PBS at 37°C for 20 min. The primary antibody CXCR2 rabbit PAb (1:100 dilution) was applied in the slides at 4°C overnight. And then the slides were washed 3 times with PBS and incubated with the second antibody Alexa Fluor 555 donkey anti-rabbit (1:200 dilution) (Beyotime Institute of Biotechnology, Nantong, China) at 37°C for 45 min. Finally, the slides were washed 3 times with PBS and counterstained with DAPI and observed with a Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss, Inc.).
Axiovert 200m epifluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert 200M is an epifluorescence microscope manufactured by Zeiss. It is designed to perform fluorescence imaging and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrm camera, by Zeiss (3 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Southern Biotech (2 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (2 mentions)
F4 80 monoclonal antibody, by Abcam (2 mentions)
Plan apochromat 63 1.4 na oil objective, by Zeiss (2 mentions)
Aqua poly mount, by Polysciences (2 mentions)
Glycergel, by Agilent Technologies (2 mentions)
Dihydroethidium dhe, by Merck Group (1 mentions)
Apochromat 63 oil immersion objective, by Zeiss (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Nunc lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
Axiovision software package, by Zeiss (1 mentions)
8 well chamber slide, by BD (1 mentions)
Axiovert 200 m system, by Zeiss (1 mentions)
Axiovision extended focus module, by Zeiss (1 mentions)
Matlab, by MathWorks (1 mentions)
X cite exacte, by Excelitas (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti claudin 3, by Thermo Fisher Scientific (1 mentions)
37 protocols using axiovert 200m epifluorescence microscope
1
CXCR2 Immunofluorescence Assay Protocol
2
Macrophage Reactive Oxygen Imaging
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RAW 264.7 macrophages were cultured on glass coverslips in 24-well plates in complete medium. Macrophages were infected with Listeria strains as described above. Cells were washed once with PBS and stained for 45 min with 5 µM dihydroethidium (DHE; Sigma) in PBS. Cells were washed and fixed with 4% paraformaldehyde (PFA) in PBS. Samples were mounted on glass coverslips with Fluoromount medium (Electron Microscopy Sciences) and were observed with a Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss, Inc.) connected to a charge-coupled device (CCD) camera, using DAPI (4′,6-diamidino-2-phenylindole) and rhodamine filters. Images were acquired with an apochromat 63× oil immersion objective (Carl Zeiss, Inc.) and processed with Metamorph software (Universal Imaging).
3
Inhibition of VEGF-HSPG Interactions
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To remove high capacity VEGF-binding sites associated to HSPGs whose interaction with VEGF could mask the specific interaction of the growth factor with VEGFR2, cells were treated with chlorate to inhibit sulfation of HS chains as described [28 (link)]. Chlorate-treated ECD-VEGFR2-EYFP GM7373 cells were incubated for 90 min at 4 °C with VEGF (75 ng/ml) and UniPR1331 (30 μM) in Hanks’ balanced salt solution (HBSS) with calcium and magnesium, washed with PBS, and fixed in 4% paraformaldehyde. Immunofluorescence analysis was performed using an anti-VEGF antibody and Alexa Fluor 594 anti-mouse IgG. Cells were photographed using a Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss, Gottingen, Germany).
4
Microscopy of Cable Bacteria Across Oxic-Anoxic Interface
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A glass slide sandwich system was inserted into the sediment to observe cable bacterium filaments across the oxic-anoxic interface. To this end, two microscopy slides were pressed against each other and ASW was added in between. These “double slides” were then inserted half-way into the sediment of enrichment cultures (sieved 350 μm mesh) such that the longer edge was parallel to the sediment surface. This arrangement allowed the development of opposing gradients of sulfide and oxygen within the layer of ASW between the slides. After several weeks, when numerous cable bacteria filaments were observed between the slides, the slides were carefully separated, the bacteria were stained with the general DNA stain 4′,6-diamidino-2-phenylindole (DAPI), and a coverslip was placed on top and sealed with a nail polish. The stained filaments were then imaged using a Zeiss Axiovert 200M epifluorescence microscope (Carl Zeiss, Göttingen, Germany) equipped with the Zeiss filter set 02 (excitation G365, beamsplitter: BS395; emission LP420) and filter set 09 (excitation: BP450-490, beamsplitter: FT 510, emission: LP 515).
5
Transient transfection of Fam20C-GFP in HEK293 and MC3T3 cells
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The 20.6 kb Fam20C-GFP plasmid was transiently transfected into HEK293 and MC3T3 cells using Lipofectamine 2000 (Invitrogen, Grand island, NY, USA). In brief, these two cell lines were seeded onto 10 cm dishes (80% confluent). Ten microliters of Lipofectamine with 2 µg construct was added to serum starved cells. The media was changed after 4 h with normal serum medium. As a control, the cells were transfected with the original empty pBluescript SK plasmid with a cytomegalovirus (CMV) promoter (Agilent Technologies, Santa Clara, CA, USA). The GFP signal was observed and imaged using a GFP filter cube on a Zeiss Axiovert 200M epifluorescence microscope (Zeiss, Jena, Germany) 48 h later.
6
Epifluorescence Microscopy of PSCA-Targeted CD8+ T Cells
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For epifluorescence microscopy 5×103 PC3-PSCA tumor cells were cultivated with 5×104 CD8+ T cells in the presence or absence of 30 pmol/ml scBsTaFv CD8-PSCA(MB1) in Nunc Lab-Tek Chamber Slides (ThermoFisher Scientific, Schwerte, Germany) for 22 h. Thereafter, medium was discarded and cells were fixed with 90% methanol for 10 minutes at −20°C and blocked with 5% human serum in 1× PBS for 15 min at 4°C. Finally, the scBsTaFv CD8-PSCA(MB1) was detected with anti-myc/FITC mAb (Miltenyi Biotec). Samples were washed and covered with DAPI-containing cover medium AKLIDES (Medipan, Dahewitz, Germany) and cover slides. Microscopic images were taken with a Zeiss Axiovert 200M epifluorescence microscope and edited with Axiovision software package (Zeiss, Jena, Germany).
7
Double Immunofluorescence Staining of Liver Sections
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Immunofluorescence staining was performed as described before.4 , 28 Briefly, for double‐staining, monoclonal F4/80 antibody (Abcam, Cambridge, UK; dilution 1:10) was co‐incubated with polyclonal antibody directed against cytokeratin 19 (CK19) (Abcam; dilution 1:50). Liver cryosections of 5 μm thickness were fixed with cold acetone/methanol, washed with PBS, and incubated with blocking medium (0.1% BSA, 10% FCS in PBS) for 1 hour. Sections were incubated with primary antibody at 4°C overnight. Non‐immune serum served as negative control. Secondary antibodies were purchased from Molecular Probes (Leiden, The Netherlands; dilution 1:400). DAPI (4′,6‐diamidino‐2‐phenylindole; Southern Biotech, Birmingham, USA) was used for nuclear counterstaining. The stained sections were investigated with an Axiovert 200M epifluorescence microscope (Zeiss, Jena, Germany).
8
SARS-CoV-2 Immunofluorescence Staining
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Uninfected cells were seeded (5 × 104 cells per well) in 8-well chamber slides (Becton–Dickinson, Franklin Lakes, NJ, USA) and then infected as described above. After infection, cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with 0.1% Triton X100 in PBS, and saturated with 3% bovine serum albumin (BSA), 0.1% Tween 20 in PBS. For staining, cells were incubated overnight with a human serum containing IgG to SARS-CoV-2 (1:1000 dilution), with an anti-SARS-CoV-2 spike (S) glycoprotein monoclonal antibody (1:1000 dilution; abcam, clone 1A9, Cambridge, UK), anti-SARS-CoV-2 nucleocapsid (NP) monoclonal antibody (1:1000 dilution; abcam, clone 6H3), anti-SARS-CoV-2 envelope (E) polyclonal antibody (1:1000 dilution; abcam) followed by Alexa Fluor 488-conjugated anti-human, mouse or rabbit IgG (Thermo Fisher Scientific). Nuclei were counterstained with 4′,6-diamidino,2-phenylindole (DAPI, Merck, Darmstadt, Germany). Cells were photographed under a Zeiss Axiovert 200 M epifluorescence microscope equipped with a Plan-Apochromat 40x or 63x/1.4 NA oil objective. Z-stack images acquired using the ApoTome imaging system were elaborated through the AxioVision Extended Focus module (Zeiss Axiovert 200 M system).
9
Fluorescence Microscopy Imaging Protocol
10
Immunofluorescence Imaging of Claudin-3
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Snap frozen tissue sections were fixed in 3% paraformaldehyde/2% sucrose in PBS, permeabilized with 0.5% Triton-X100, and saturated with goat serum in PBS. Samples were incubated with anti-claudin3 (Invitrogen) or with IgGH6 anti-claudin3 [13 (link)] over-night at 4°C followed by Alexa Fluor 594 anti-rabbit IgG (Molecular Probes, Eugene, OR). Cells membranes were stained with WGA lectin-Alexa Fluor 488 (Molecular Probes, Eugene, OR) and nuclei were counterstained with 4’,6-diamidino,2-phenylindole (DAPI, Sigma). Samples were analyzed using Zeiss Axiovert 200M epifluorescence microscope equipped with Apotome system, Plan-Neofluar 20x/0.5 NA and Plan-Apochromat 63x/1.4 NA oil objectives. Z-stack images were elaborated with AxioVision Inside4D module (Carl Zeiss) [24 (link)].
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