Eight-micron cryosections were permeabalised and blocked in 10% goat serum in TRIS-buffered saline (TBS) 0.1% Triton X-100 for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C (ESM Table 3 ). Appropriate secondary antibodies conjugated with Alexa 488/594/647 fluorophores from Life Technologies (Carlsbad, CA, USA) were applied for 1 h at room temperature (1:1000). Digital images of the cryosections mounted with fluorescence mounting medium (Dako, Glostrup, Denmark) were acquired with a Zeiss LSM 800 Airyscan confocal laser scanning microscope.
Lsm 800 airyscan confocal laser scanning microscope
Manufactured by Zeiss
Sourced in Denmark
The LSM 800 Airyscan confocal laser scanning microscope is a high-resolution imaging system from Zeiss. It utilizes a specialized detector array to capture high-quality, high-resolution images with improved signal-to-noise ratio compared to conventional confocal microscopes.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Texas red dextran, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Citrullinated h3, by Cayman Chemical (1 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
6 protocols using lsm 800 airyscan confocal laser scanning microscope
1
Immunofluorescence Staining of Cryosections
2
Visualization of Cell Membrane Dynamics
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Eight million (8 × 106) 16HBE cells were seeded into each well of a collagen-coated, six-well plate and were grown at 37°C for ~24 h in a CO2-jacketed incubator until they reached 100% confluence. After treatment, cells were washed in serum-free EMEM and detached from the microtiter plate by trypsin-EDTA solution (MilliporeSigma). After inactivating the trypsin with media containing 10% fetal bovine serum, cells were concentrated via centrifugation and transferred onto a glass side with a 3% noble agar pad. These slides were visualized under an LSM800 AiryScan confocal laser scanning microscope (Zeiss). Pyoverdine fluorescence was visualized via a 405 nm laser line using the channel conditions for Pacific Blue. Dextran-Texas Red (Invitrogen) fluorescence was visualized via a 561 nm laser line using channel conditions for Texas Red. CellMask Deep Red plasma membrane stain (Invitrogen) fluorescence was visualized via a 640 nm laser line using channel conditions for Alex Fluor 660.
3
Liver Histological Analysis of Mice
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Mice were sacrificed by cervical dislocation and livers were perfused with PBS via the inferior vena cava. Preparation of paraffin-embedded liver sections and staining with hematoxylin and eosin (H&E) or picrosirius red were performed as described previously34 (link). The sections were treated for antigen retrieval by high-temperature heating in Tris–EDTA buffer and stained using primary antibody against αSMA and secondary anti-rabbit Alexa 488 antibodies.
Immunohistochemical staining of frozen tissue was performed as previously described54 (link). Eight-micrometer frozen sections were stained overnight with primary anti-IL-33, anti-PDGFRα or anti-CD45 antibodies at 4 °C followed by incubation with appropriate secondary antibodies conjugated with Alexa 488/594 fluorophores from Life Technologies (Thermo Fisher Scientific, Darmstadt, Germany) for 1 h at room temperature (1:500). Digital images of the sections mounted with fluorescence mounting medium (Dako, Glostrup, Denmark) were acquired with a Zeiss LSM 800 Airyscan confocal laser-scanning microscope.
Immunohistochemical staining of frozen tissue was performed as previously described54 (link). Eight-micrometer frozen sections were stained overnight with primary anti-IL-33, anti-PDGFRα or anti-CD45 antibodies at 4 °C followed by incubation with appropriate secondary antibodies conjugated with Alexa 488/594 fluorophores from Life Technologies (Thermo Fisher Scientific, Darmstadt, Germany) for 1 h at room temperature (1:500). Digital images of the sections mounted with fluorescence mounting medium (Dako, Glostrup, Denmark) were acquired with a Zeiss LSM 800 Airyscan confocal laser-scanning microscope.
4
Citrullinated Histone H3 Detection
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Paraffin-embedded lung sections were subjected to deparaffinization followed by heat induced antigen retrieval in 10 mM sodium citrate buffer (pH 6.0). Sections were blocked with 10% goat serum in 1x PBS for 1 hour. Primary antibody staining was performed for citrullinated H3 (Cayman Chemical, Cat. No. 17939, 1:50) overnight at 4°C. Slides were then incubated with Alexa Fluor 633 anti-mouse IgG secondary antibody (Thermo Fisher Scientific, Cat. No. A21052, 1:1000) for 90 mins at room temperature. Images were taken at 20x objective using a Zeiss LSM 800 Airyscan laser scanning confocal microscope.
5
Immunofluorescence Staining of Cells and Tissues
6
Immunofluorescence Labeling of Cultured Cells
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Cultured cells were fixed with cold 4% PFA for 20 min (or with ice-cold MeOH for 5 min, in the case of astrocytic cultures), washed with PBS/glycine, and permeabilized with 0.2% Triton-X100 for 30 min at room temperature.
Unspecific binding sites were blocked with 3% bovine serum albumin (BSA, MP Biomedicals) for 60 min, and labelling with primary antibodies was performed overnight at 4°C. After incubation, cells were extensively washed with PBS/Triton-X100 and incubated with appropriate secondary antibodies at room temperature for 2 hr. After nuclear staining with Hoechst 33342 or DAPI, samples were mounted and images were acquired in a ZEISS LSM 800-AiryScan laser scanning confocal microscope provided with a Zen Blue Edition V2.6 program or a Zeiss LSM880 confocal microscope provided with a Zen Black Edition V2.3 program.
Unspecific binding sites were blocked with 3% bovine serum albumin (BSA, MP Biomedicals) for 60 min, and labelling with primary antibodies was performed overnight at 4°C. After incubation, cells were extensively washed with PBS/Triton-X100 and incubated with appropriate secondary antibodies at room temperature for 2 hr. After nuclear staining with Hoechst 33342 or DAPI, samples were mounted and images were acquired in a ZEISS LSM 800-AiryScan laser scanning confocal microscope provided with a Zen Blue Edition V2.6 program or a Zeiss LSM880 confocal microscope provided with a Zen Black Edition V2.3 program.
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