Fluorescein labeled secondary antibodies

Immunofluorescence was performed as previously described (Qi et al. 2017 (link)). Briefly, the antrum tissues from human, mouse, rat and pig were washed in cold PBS and were fixed in 4% paraformaldehyde for overnight at room temperature. Then paraffin-embedded antrum sections were de-paraffinized in xylene and dehydrated by a graded alcohol series, followed by antigen retrieval. Next, after washing by PBS for 3 times, the sections were blocked and permeabilized by 0.3% Triton X-100 and 5% BSA for 1 h at room temperature. Then, the sections were incubated with primary antibodies overnight at 4 °C. The fluorescein-labeled secondary antibodies (Life Technologies, 1:300) and 4′, 6-diamidino-2-phenylindole (DAPI) were added for 1 h at room temperature next day. The images were acquired from Olympus FV3000 Laser Scanning Microscope.

For immunofluorescence, intestine and organoids were fixed for overnight with 4% formaldehyde at room temperature. Then paraffin intestine or organoid sections were de-paraffinized in isopropanol and dehydrated by a graded alcohol series, followed by antigen retrieval. Next, the section was permeabilized for 10 min in PBS with 0.1% Triton X-100 at room temperature. Then the sections were blocked in 5% BSA/0.1% Triton-X/PBS (blocking buffer) for 1 h at room temperature followed by incubating with the primary antibody at 4°C overnight. The fluorescein-labeled secondary antibodies (1:300, Life Technologies) were added for 1 h at room temperature. Samples were visualized by an Olympus FV1200 Laser Scanning Microscope.
For immunohistochemistry, the paraffin section was prepared as immunofluorescence. Then endogenous peroxidase was quenched by H₂O₂. Next, the sections were blocked in blocking buffer for 30 min and incubated overnight with primary antibody at 4°C. Horseradish peroxidase-conjugated secondary antibody (Invitrogen, 1:200) was added for 2 h at room temperature followed by developing with substrate DAB. Then sections were counterstained with hematoxylin and eosin, dehydrated and mounted with resinene.

Immunofluorescence was performed as previously described (Qi et al., 2017 (link)). Briefly, the ileum tissues from mouse, rat, pig, macaque and human were washed in cold PBS and were fixed in 4% paraformaldehyde for overnight at room temperature. Then paraffin-embedded ileum sections were de-paraffinized in isopropanol and dehydrated by a graded alcohol series, followed by antigen retrieval. Next, the sections were washed by PBS for 3 times and permeabilized by 0.1% Triton X-100 for 15 min at room temperature. Then, the sections were blocked with PBT solution (3% BSA and 0.01% Triton X-100 in PBS) for 1 h at room temperature, followed by incubating with primary antibodies overnight at 4 °C. The fluorescein-labeled secondary antibodies (Life Technologies, 1:300) and 4′, 6-diamidino-2-phenylindole (DAPI) were added for 1 h at room temperature next day. The images were acquired from Olympus FV3000 Laser Scanning Microscope.

The cryo-sections of mouse LV and cultured mouse primary cells were used for immunofluorescence staining in this study. The hearts of the mice were freshly embedded in OCT and sectioned at the thickness of 7 μm. The heart sections or cells were first fixed in 4% PFA for 15 minutes and washed with PBS for 10 minutes. Then, the samples were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. Nonspecific binding was reduced by the incubation of 10% rabbit sera diluted in PBST for 20 minutes at room temperature. The samples were then incubated with antibodies against CD68 (Bio-Rad AbD Serotec, MCA1957), rabbit MCP-1 (Abcam, ab7202), IL-6 (Bio-Rad AbD Serotec, MCA1490), α-SMA (Abcam, ab5694), collagen I (Abcam, ab34710), ER-TR7 (Novus Biologicals, NB100-64932), Myh7 (Abcam,ab50967), or Ki67 (Abcam, ab15580) at 4°C for 12 to 15 hours. The sections or cells were rinsed with PBS and incubated with fluorescein-labeled secondary antibodies (Life Technologies). The nuclei were stained by mounting the slides with DAPI medium (Vector Laboratories). TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche Applied Science) as previously described (14 (link)). Images were acquired using Nikon fluorescence microscopy.

At the indicated time points post transfection or bacterial infection, cells were fixed for 10 min with 4% paraformaldehyde in PBS and permeabilized for 15 min with 0.2% Triton X‐100 in PBS. After the blockade of nonspecific binding by incubation of cells for 30 min with 2% bovine serum albumin in PBS, coverslips were incubated with the appropriate primary antibodies and subsequently with fluorescein‐labeled secondary antibodies (Thermo Fisher Scientific). Confocal fluorescence images were acquired under the confocal microscope (Spinning Disc; Leica). All image data shown are representative of at least three randomly selected fields.

Immunofluorescence labeling was conducted according to our standard protocols (Meng et al., 2020 (link)). At the indicated time post-AE-treatment, cells were fixed with 4% PFA for 10 min in PBS and permeabilized for 15 min with 0.2% Triton X-100 in PBS. After blockade of nonspecific binding by incubation of cells for 30 min with 2% bovine serum albumin (BSA) in PBS, samples were incubated with the appropriate primary antibodies and subsequently incubated with fluorescein-labeled secondary antibodies (ThermoFisher). Confocal fluorescence images were acquired at the confocal microscope (Olympus). All image data shown are representative of randomly selected fields from at least three replicates.