PMSS1 gland colonization was assessed as previously described (8 (link)) with the following modifications. One third of the stomach was embedded in 4% agarose in 1× phosphate-buffered saline (PBS, Gibco) and cut into 200 μm thick longitudinal sections using a Leica VT1200S Vibratome. Tissue sections were then permeabilized overnight at 4°C by gently rocking in blocking buffer comprising 3% bovine serum albumin (Sigma-Aldrich), 1% saponin (Sigma-Aldrich) and 1% Triton X-100 (Sigma-Aldrich) in phosphate-buffered saline (PBS). Stomachs were incubated with 1:1,000 rabbit polyclonal anti-H. pylori PMSS1 antibody (gift of Manuel Amieva, Stanford University) and 1:2,000 GS-II 488 (conjugated lectin from Griffonia simplicifolia , Fisher) for 2 h at 4°C with gentle rocking. After three 10-minute washes in PBS, samples were incubated with 1:2,000 Alexa Fluor 647 donkey anti-rabbit IgG (Invitrogen) and 1:2,000 DAPI for 2 h at room temperature with gentle rocking. After three 10-minute washes in PBS, sections were mounted onto glass slides with imaging spacers cut from Parafilm (Bemis) in ProLong Diamond Anti-Fade Reagent (Molecular Probes) and coverslips were sealed with VaLP (1:1:1 Vaseline:Lanolin:Paraffin). Stomach sections were imaged on a Zeiss LSM 780 laser-scanning confocal microscope, or with an UltraView spinning disk microscope (PerkinElmer). Z-stacks were collected to visualize H. pylori within glands and assessed in Volocity (Quorum Technologies) to enumerate H. pylori in glands based on fluorescent voxels as previously described (8 (link)).
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