Lsm700 axio examiner z 1 confocal microscope

Bacterial penetration of the inner mucus layer was scored using the fluorescence-in situ-hybridization (FISH)-, anti-Muc2- and DNA-stained sections as described previously (S2 Table) [28 (link)]. The mucus layering score, focusing solely on the layering and structure of the inner, stratified mucus layer and the goblet cell filling score [16 (link)] were applied (S3 Table and S4 Table, respectively). Sections were scored independently by two blinded individuals, except for the naive controls which were scored by one blinded individual using the Eclipse E1000 and Eclipse90i (Nikon) fluorescence microscope as well as the Axiovert 200M microscope (Zeiss) with the EXFO X-Cite 120 Fluorescence Illumination System (Olympus, Hamburg, Germany). Micrographs were obtained using the LSM700 Axio Examiner Z.1 confocal microscope (Zeiss) with the ZEN 2010 software (Zeiss).

Tissue specimens were washed in cold PBS, fixed in 10% formalin buffered saline for 2 hr, followed by incubation in 30% sucrose overnight. Tissue specimens were embedded in optimal tissue cutting medium and 6-µm-thick section were cut. Sections were rinsed in PBS followed by antigen retrieval in 0.05% citraconic anhydride (Sigma-Aldrich, St Louis, MO), permeabilized in 0.05% Triton X-100 or 0.05% saponin (TGN46) (Sigma-Aldrich, St Louis, MO), washed in PBS, and blocked in 5% goat serum (Sigma-Aldrich, St Louis, MO). Sections were incubated with primary antibodies toward EEA1, Rab7 (Cell Signaling Technology, Beverly, MA), Lysozyme, TGN46 (Thermo Fisher Scientific, Waltham, MA), Rab3D (Synaptic Systems, Goettingen, Germany), LAMP1, Calnexin (Abcam, Cambridge, United Kingdom), or VAMP-8 (gift from Prof Burton Dickey, University of Texas) at 4°C overnight, rinsed with PBS and incubated with goat anti rabbit or goat anti chicken Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA) at room temperature for 1 hr. Sections were counterstained with DAPI and imaged using an Axioskop 2 microscope, an LSM700 Axio Examiner Z1 confocal microscope, or an LSM900 with Airyscan 2 microscope (Carl Zeiss Microscopy, Thornwood, NY). Acquired images were analyzed using Zen (Carl Zeiss Microscopy, Thornwood, NY) and Imaris (Bitplane, Belfast, Great Britain) software.

The mucus penetrability assay was performed on distal colon tissue as previously described [16 (link),37 (link)]. Briefly, the colonic explants were mounted in the perfusion chamber with basolateral Kreb’s glucose solution containing 1μg/ml Calcein violet tissue stain (CellTrace Calcein Violet, Molecular Probes, Thermo Fisher) and a suspension of far red fluorescent beads (FluoSpheres Carboxylate-Modified Microspheres, 1.0 μm, red fluorescent (580/605), 2% solids, Molecular Probes, Thermo Fisher) and Kreb’s mannitol solution was added to the apical side and allowed to sediment for 5 min before it was refilled with Kreb’s mannitol solution. The distribution of the beads in the mucus was investigated by acquiring confocal images in XY stacks (320 x 320 μm) 30min after tissue mounting. These were obtained using a LSM700 Axio Examiner Z.1 confocal microscope with Plan-Apochromat x 20/1.0 DIC water objective (Zeiss) and the ZEN 2010 software (Zeiss). Average bead distance from the tissue surface was calculated by combining bead fluorescence intensity data for each z-plane above the tissue surface.