Multilabel immunofluorescence analysis was performed on a Leica DM4000B or a Zeiss LSM 710 confocal microscope. Images were acquired and minimally processed by importing them into the GNU image manipulation program GIMP and the ImageJ software. Quantification of Muc2 and UEA1 or SNA colocalization in the ileum and in the colon (n = 7 mice/group) was performed on 4–7 villi or crypts/mouse. Fluorescence was measured and expressed as the UEA1/Muc2 and SNA/Muc2 (data not shown) ratio relative to the area of the villus or the crypt.
Dm4000b
Manufactured by Zeiss
The DM4000B is a high-performance optical microscope designed for a variety of laboratory applications. It features advanced optics and versatile functionality to support various imaging and analysis needs. The core function of the DM4000B is to provide a reliable and precise platform for detailed microscopic examination of samples.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 700, by Zeiss (2 mentions)
Axiovision software, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Mabn50, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by BD (1 mentions)
Sc 17320, by Abcam (1 mentions)
Z0334, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Smz25, by Nikon (1 mentions)
Bz x700, by Leica (1 mentions)
Stellaris 5, by Leica (1 mentions)
4 protocols using dm4000b
1
Quantifying Mucin and Lectin Colocalization
2
DAPI-based Fluorescence Imaging of Cells
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Cells were prepared for fluorescence imaging. Cells harvested were washed twice in (1 mL), resuspended in DAPI (1 μM, Invitrogen, UK), and incubated in a dark chamber for 15 min at room temperature. Aliquots of 3–5 μL of the resuspended cells were deposited on slides, covered with glass slips and visualized with the blue filter (excitation wavelength 340–380 nm and emission wavelengths 450–490 nm) using a LEICA DM4000B fluorescence microscope with Zeiss AxioVision software.
3
Immunofluorescence Staining of Tissue Sections
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Tissue was processed as described above. Sections or cells were incubated in sodium citrate (10 mM, microwave preheated) for antigen retrieval, blocked with PBS containing 0.3% (v/v) Triton X-100 and 10% (v/v) normal donkey serum (1 hr, RT), and incubated with primary antibody overnight, 4°C.
Primary antibody used: Caspase3 R and D Systems AF835-SP; CTGF Abcam ab6992; GFAP DakoCytomation Z0334; GFAP BD Biosciences 556330; GFP Abcam ab6673; MPZ Abcam ab39375; Neurofilament NF DakoCytomation M0762; Olig2 Abcam ab33427; Olig2 Millipore MABN50; Periaxin Gift from Prof. Peter Brophy, University of Edinburgh, UK; SMI71 Calbiochem NE1026; Sostdc1 Abcam 99340; Sox10 Gift from Dr. Michael Wegner, Universität Erlangen-Nürnberg, Erlangen, Germany; Sox2 Santa Cruz Biotechnology sc-17320; vWF Abcam ab6994; Iba1 Wako 019–19741, MBP R@D Systems MAB42282, tRFP Evrogen AB233.
Then, sections were incubated with appropriate AF488, AF555, or AT647- conjugated secondary antibodies (1:500, ThermoFisher Scientific). Nuclei were visualized with 4’, 6’-diamidino-2-phenylindole (DAPI; 0.1 mg/ml; Sigma). Images were acquired with fluorescence microscope (Leica DM4000B) and confocal (Zeiss LSM700) microscopes.
Primary antibody used: Caspase3 R and D Systems AF835-SP; CTGF Abcam ab6992; GFAP DakoCytomation Z0334; GFAP BD Biosciences 556330; GFP Abcam ab6673; MPZ Abcam ab39375; Neurofilament NF DakoCytomation M0762; Olig2 Abcam ab33427; Olig2 Millipore MABN50; Periaxin Gift from Prof. Peter Brophy, University of Edinburgh, UK; SMI71 Calbiochem NE1026; Sostdc1 Abcam 99340; Sox10 Gift from Dr. Michael Wegner, Universität Erlangen-Nürnberg, Erlangen, Germany; Sox2 Santa Cruz Biotechnology sc-17320; vWF Abcam ab6994; Iba1 Wako 019–19741, MBP R@D Systems MAB42282, tRFP Evrogen AB233.
Then, sections were incubated with appropriate AF488, AF555, or AT647- conjugated secondary antibodies (1:500, ThermoFisher Scientific). Nuclei were visualized with 4’, 6’-diamidino-2-phenylindole (DAPI; 0.1 mg/ml; Sigma). Images were acquired with fluorescence microscope (Leica DM4000B) and confocal (Zeiss LSM700) microscopes.
4
Multimodal Imaging of Zebrafish Embryos
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Embryos were imaged at 12 hpf, 24 hpf, 48 hpf, 72 hpf, and 5 dpf on Nikon SMZ25 fluorescent stereomicroscope. Images of whole mount immune-stained zebrafish were taken on a Keyence BZ-X700 fluorescent microscope and Leica STELLARIS 5. Slides were imaged on a Keyence BZ-X700 fluorescent microscope, Leica DM4000B and Zeiss LSN710.
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