C57BL/6J female mice were fasted overnight and AvrA-eGFP nanoparticles (12 μg eGFP) were injected intrarectally the following day. After 5 h, mice were sacrificed and colon tissue was removed. Distal colon was fixed in 4% paraformaldehyde and embedded in Tissue-Tek O.C.T. Compound. Twenty micrometer frozen sections were immunostained with anti-β-catenin and anti-GFP antibodies, and mucosal uptake of eGFP particles were imaged using a Zeiss LSM 510 confocal laser scanning system.
Lsm 510 confocal laser scanning system
Manufactured by Zeiss
The LSM 510 is a confocal laser scanning microscope system manufactured by Zeiss. It is designed to provide high-resolution optical sectioning and imaging of samples. The system utilizes a laser source, scanning mirrors, and a photomultiplier tube detector to generate detailed images of specimens.
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2 protocols using lsm 510 confocal laser scanning system
1
Visualizing Nanoparticle Uptake in Colon Tissue
2
Assessing Monolayer Integrity via ZO-1 Immunostaining
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Following some experiments, bEnd.3 monolayers were immunostained for the tight junction protein ZO-1 to assess monolayer integrity. After 60 min of convective flux, control (no DCS) and DC-stimulated monolayers were washed with PBS and fixed in 1% paraformaldehyde (PFA; Polysciences Inc., Warrington, PA) for 10 min. Monolayers were then permeabilized with 0.2% Triton X-100 for 10 min, blocked with 10% goat serum and 0.1% Triton X-100 for 1 hr, and incubated with primary rabbit anti-ZO-1 antibody (1:200; Invitrogen, Rockford, IL) overnight at 4 °C. After washing 5X with PBS, ZO-1 was detected with AF555 goat anti-rabbit IgG secondary antibody (1:500; Life Technologies Inc, Eugene, OR) for 60 min at room temperature. After washing 4X with PBS, monolayers were counterstained with DAPI nuclear stain and mounted with Fluoromount (Southern Biotech, Birmingham, AL). Imaging was conducted in a Zeiss LSM 510 Confocal Laser Scanning system at 40X/oil magnification.
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