Prolong glass

Monolayers of primary gastric epithelial cells derived from C57BL/6 wild-type and Sting^−/− mice were infected for 24 hours with H. pylori strains J166 or PMSS1. After infection, monolayers were subjected to immunofluorescence staining as previously described.^{17 (link),50 (link)} Briefly, cells were fixed with 10% neutral-buffered formalin (Azer Scientific), permeabilized with Triton X-100 (Promega), and then blocked with Dako Protein Block Solution (Agilent) for 1 hour. Samples were incubated with anti-TRIM30 rabbit antibody #NBP2-41087 (Novus Biologicals; 1:50) overnight at 4°C before detection with Alexa-fluor secondary antibody (Invitrogen). Nuclei were detected using Hoescht (Invitrogen). Slides were mounted using ProLong Glass (Invitrogen), and images were acquired in an Olympus FV-1000 confocal microscope. Experiments were performed in part through the use of the Vanderbilt Cell Imaging Shared Resource (CISR).

The Duolink^® PLA (Merck) was used to detect close proximity of endogenous ALIX with CHMP4, Rab11, TSG101, KIF5B or KLC1. The assay is based on oligonucleotide-conjugated PLA probes, containing secondary antibodies directed against primary antibodies against the proteins of interest. Annealing of the probes occurs when the target proteins are in close proximity (< 40 nm), which then initiates the amplification. The amplicons can be detected by fluorescence microscopy in a quantifiable manner. For this assay, Hela K cells were seeded on coverslips in six-well plates and the PLA experiments were performed the next day with subconfluent cells. Cells were washed, fixed and permeabilized as described in the section above. Antibodies against ALIX (1:100, BioLegend #634502), CHMP4B (1:500, [16 (link)]), Rab11 (1:100, Invitrogen #71-5300), TSG101 (1:100, Sigma #HPA006161), KIF5B (1:100, Abcam #ab151558), KLC1 (1:100, Santa Cruz #sc25735) and tubulin (1:200, Cytoskeleton #ATN02) were used as primary antibodies, and the assays were performed as described in the manufacturer’s manual. Slides were mounted with ProLong Glass (Invitrogen) and samples were observed by fluorescence microscopy with a Nikon ECLIPSE Ti2 spinning-disk microscope (number of experiments: ALIX + CHMP4B = 4, ALIX + KIF5B = 4, ALIX + KLC1 = 4, ALIX + Rab11 = 2, ALIX + TSG101 = 2).

Paraformaldehyde-fixed tissue sections (30 μm thick) were obtained from the Cleveland Clinic rapid autopsy program. Antigen retrieval was performed using 10 μM citrate buffer and boiling briefly. Free-floating sections were permeabilized with 2% Triton-X100 in PBS for 30 min then blocked with 5% normal goat serum and 0.3% Triton-X100 for 1 h. Sections were then incubated in primary antibody against major histocompatibility complex class II (MHCII; 1:50, Abcam), myelin basic protein (MBP; 1:200, Abcam), glial fibrillary acidic protein (GFAP; 1:50, Invitrogen), and EDC4 (1:40, Abcam) for 3 days. Sections were then washed in PBS with 0.3% Triton-X100 three times and incubated with secondary antibodies (Invitrogen) conjugated to AlexaFluor 405, 488, 594, or 647 for 1 h at room temperature. Slides were then washed three times and treated with TrueBlack lipofuscin quencher (Biotium) per manufacturer’s instructions. Sections were mounted using Prolong Glass (Invitrogen) and stored at -20°C until imaged using a Zeiss LSM800 confocal microscope. Cell bodies filled with EDC4 were counted by a blinded observer.

Sample preparation for Airyscan confocal immunofluorescence microscopy. Mice 6 weeks of age were transcardially perfused with 80 mM PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl₂, 4% paraformaldehyde). Eyes were enucleated, and after the cornea was removed, they were immersion fixed overnight at 4 ^oC. After removal of lens, the eyecups were flash-frozen in optimal cutting temperature using liquid nitrogen. 8 μm cryosections were collected and stained for rhodamine wheat germ agglutinin (Vector RL-1022) and phalloidin conjugated to Atto647N (Sigma 65906) or cofilin (CST 5175) and phalloidin conjugated to Atto647N (Sigma 65906). The sections were then mounted in Prolong Glass (Invitrogen P36980). Sections were imaged on a Zeiss LSM 880 Airyscan Fast Confocal Microscope using a 63x objective. Z-stacks were first processed in Zeiss ZenBlue software for Airyscan processing, then colour processing and analysis were performed in Fiji/ImageJ. Actin puncta quantification was performed by taking a 0.35 μm ROI around each actin puncta that was at the base of an outer segment, slice by slice, on each z-stack. Then the averaged relative integrated density of three background ROI’s were subtracted from each relative integrated density measurements from the actin ROIs. These measurements were then plotted in Prism software, where Students t-test statistical analysis was performed.

Cells were directly grown on glass coverslips (0.17 mm thickness, 1.5 H high performance; Marienfeld Superior) placed in 24-well plates. Cells were fixed with 4% paraformaldehyde (20 min; RT), permeabilized with methanol (10 min RT) and blocked with 5% bovine serum albumin (5% BSA diluted in phosphate-buffered saline [PBS]-Triton 0.1%). Primary antibodies of interest were incubated overnight at 4°C. Secondary antibodies conjugated to Alexa fluorophores were incubated 1 hr RT in the dark. The following conjugated secondary antibodies (Invitrogen) were used: goat anti-rabbit IgG H+L (Alexa-488 or -647); donkey anti-mouse IgG H+L (Alexa-555, -561, -633, or -647). After incubation, samples were washed with PBS-T (x3) and nuclei were stained with Hoechst (25 µg/ml diluted in 5% BSA-PBS-T; 1 hr RT in the dark). Five PBS-T washes and a final MiliQ water were performed. Coverslips were mounted using Pro-long glass (Invitrogen). Preparations were maintained 24–48 hr in the dark at RT and then stored at 4°C up to image acquisition.

Aortic sinuses were fixed in 4% paraformaldehyde (Fisher) for 10 min at room temperature and washed in PBS. The sections were blocked and permeabilized in 10% normal horse serum (Vector) and 0.1% Triton X-100 (Fisher) for 30 min at room temperature. Primary antibodies were diluted in 1% normal horse serum and incubated for 2 h at room temperature or overnight at 4°C. Secondary antibodies were incubated at room temperature for 1 h 30 min in 1% normal horse serum. Nuclei were stained using DAPI (Invitrogen) for 20 min at room temperature. Slides were mounted using Prolong Glass (Invitrogen) and #1.5 coverslips (Ibidi). The aortic sinuses were imaged using a Zeiss AxioObserver Z1 at 20x (NA 0.8). Images were quantified using FIJI (NIH) and blinded with respect to sex and treatment groups. The antibodies used are listed in Supplementary Table S1.