Samples were imaged using the Zeiss Axio Scan.Z1 slide scanner or Zeiss LSM900 Confocal (Zeiss, Toronto, Canada). Crypt length and area measurements were collected using the Zen Blue Software (Zeiss). Images were processed using Adobe Photoshop (San Jose, CA). Antibody-positive cells were quantified as the total number of labeled cells or nuclei/crypt, area (for measurements combining crypts and lesions), Villin positive epithelial cells, or CD45 positive blood cells. Ten crypts were sampled per animal for each colon region (proximal and distal) for quantification of ChgA, Nr1d1, CD45, pHH3, cleaved caspase-3 and Cldn1. Regions were averaged together for results shown if no significant differences were observed. The Villin/Nr1d1 and Cldn1 stained tissue were scanned using the Zeiss LSM900 Confocal while all remaining samples were imaged using the Axio Scan.Z1.
Lsm 900 confocal
Manufactured by Zeiss
Sourced in Germany, Canada
The LSM 900 confocal is a high-performance laser scanning microscope designed for advanced imaging applications. It features a versatile optical system, high-resolution imaging capabilities, and customizable configuration options to meet the needs of various research and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Plan apochromat 20 0.8 na objective, by Zeiss (1 mentions)
Axio observer z1 7, by Zeiss (1 mentions)
Lsm 800 confocal, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Zen microscopy software, by Zeiss (1 mentions)
5 protocols using lsm 900 confocal
1
Automated Colon Crypt Analysis
2
Confocal Microscopy Analysis of Coated Wood
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One beech wood sample spray coated with EOL-TC was investigated using a Zeiss LSM 900 confocal laser scanning microscope equipped (Oberkochen, Germany)on Axio Observer Z1/7 inverted stand, with diode lasers with 405 nm, 488 nm, 561 nm, and 640 nm wavelengths, and using a plan-apochromat 20×/0.8NA objective (Carl Zeiss S.p.A., Milan, Italy). The measurements were conducted in a measurement area of 319.45 × 319.45 µm using fluorescence excitation for the specific illumination of lignin (553 nm) and carbohydrate (401 nm) moieties on the coated wood sample surface. The emission wavelengths were 568 nm for lignin and 422 nm for cellulose.
3
Multimodal Microscopy Imaging of C. elegans
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Widefield fluorescence and DIC imaging were performed on a Zeiss Axioplan M2 imager as described. Conventional confocal imaging and FRAP were performed on a Zeiss LSM800 confocal. Airyscan imaging was performed on a Zeiss LSM900 confocal. 3D SIM was performed on a Deltavision OMX microscope as described, using levamisole immobilization (Adams et al., 2023 (link)). TIRF imaging was performed on the OMX SR microscope platform (Cytiva) in TIRF mode using an Olympus ApoN 60x/1.49 objective (APON60XOTIRF). The penetration depth of the evanescent wave was controlled via OMX software (OMX Acquire) and set to maximal stringency, i.e. the shallowest angle prior to loss of signal, yielding z resolutions < 100 nm.
For imaging of EPIC::mNG strains in dauer stage, dauer larvae were generated by starvation. For imaging of adults, animals were aged at least 24 h from mid L4 stage.
HaloTag JF549 ligand staining for visualization of BLI-2::HT in SIM was performed as described (Adams et al., 2023 (link)).
EPIC puncta distribution was quantitated from single focal planes of Airyscan processed images of adults 24 h post L4 stage. To measure puncta spacing we drew 10–15 μm line scans along furrow-flanking rows of puncta and counted peaks. To measure puncta density and % area we used a brightness threshold of 56–70 and a particle size range of 0.001–10 μm2, in 2–3 ROIs (100–400 μm2 each) per image.
For imaging of EPIC::mNG strains in dauer stage, dauer larvae were generated by starvation. For imaging of adults, animals were aged at least 24 h from mid L4 stage.
HaloTag JF549 ligand staining for visualization of BLI-2::HT in SIM was performed as described (Adams et al., 2023 (link)).
EPIC puncta distribution was quantitated from single focal planes of Airyscan processed images of adults 24 h post L4 stage. To measure puncta spacing we drew 10–15 μm line scans along furrow-flanking rows of puncta and counted peaks. To measure puncta density and % area we used a brightness threshold of 56–70 and a particle size range of 0.001–10 μm2, in 2–3 ROIs (100–400 μm2 each) per image.
4
Detailed Immunofluorescence Staining Protocol
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Immunofluorescence staining was performed as previously described (Wang et al., 2020 (link)). Murine back skins were embedded in Optimal Cutting Temperature (O.C.T) compound on dry ice and stored at −80°C. 16 μm skin frozen sections were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.4% Triton X-100 (Sigma-Aldrich) for 30 min, blocked in 10% donkey serum for 1 h, and incubated with primary antibodies at 4°C overnight. For 3D reconstruction analysis, the 80 μm skin sections were applied. After washing with PBS, the sections were incubated for 1 h with Alexa-488-conjugated secondary antibodies/Alexa-568-conjugated secondary antibodies (Jackson Immuno Research), and then the nucleus were counterstained with Hoechst 333 (Invitrogen). Images were captured by Leica 910 confocal or Zeiss LSM 900 confocal. Antibodies used for immunofluorescence staining were listed in Table S6 .
5
Visualization of Bacterial and Cyanobacterial Cells
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E. coli cells were visualized using acridine orange staining. Sterilized slides were fixed with 95% ethanol for 2 min, excess ethanol was drained, and the slides were allowed to air dry. Bacterial culture was placed onto a slide, dried, and briefly fixed in the flame of a burner. The slide was flooded with acridine orange stain for 2 min, rinsed thoroughly with tap water, and allowed to air dry. Cell pictures were taken on a ZEISS LSM 900 confocal laser scanning microscope (Carl Zeiss) with a 63× oil immersion objective and with 100 μm confocal pinhole aperture. Cell pictures of S. elongatus were taken similarly but using chlorophyll autofluorescence, which was excited at 488 nm and recorded using a 650-nm long-pass filter. The obtained pictures were processed using the ZEN Microscopy software (Carl Zeiss).
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