Tcs sp8 inverted fluorescence microscope

Sections of frozen ileum tissue (7 mm) were prepared and fixed with 4% PFA. Goat serum was added for 1 hour at room temperature, then the primary antibodies were applied overnight in a wet chamber at 4°C. Following the washing with PBS, the sections were incubated for 1 hour at room temperature with secondary antibodies and mounted them with VECTASHIELD Antifade Mounting Medium with DAPI to stain the nucleus. Using a Leica TCS SP8 Inverted Fluorescence Microscope (Leica Microsystems), immunofluorescent images were acquired. ImageJ software (National Institutes of Health, Bethesda, MD) was used to calculate the average fluorescence intensity of IgA and IL-10, and the number of CD11c clusters per square millimeter.
The primary antibodies and their concentrations were as follows: anti-IL-10 (bs-0698r, Bioss, China) (1:100), anti-IgA (MARA-1, Origene, USA) (1:100), and anti-CD11c (orb621157, Biorbyt, Cambridge, UK) (1:50). The secondary antibodies and their concentrations were as follows: goat anti-mouse IgG H&L-Cy3 (GB21301, Servicebio, China) (1:500), goat anti-rabbit IgG H&L-Cy3 (GB21303, Servicebio, China) (1:500), and goat anti-mouse IgG H&L-FITC (GB22301, Servicebio, China) (1:500).

As described in pervious study [38 (link)], after treatment, fixed and permeabilized gastric epithelial cell or gastric mucosa tissue slides were incubated with 2% BSA in PBS/0.05% Triton X-100 for 30 min. the slips were incubated with the primary antibody overnight at 4 °C, followed by incubation with Alexa-488- or Alexa 594- conjugated secondary antibodies for 1 h at room temperature. The coverslips were mounted onto glass slides with Prolong Gold Antifade reagent (after staining the nuclei with DAPI), and stained cells were imaged with a Leica TCS SP8 Inverted Fluorescence Microscope (Leica Microsystems). Post-acquisition processing (brightness, opacity, contrast, and color balance) was applied to the entire image and accurately reflected the results of the original image. For statistical analysis, as described in our previous work [39 (link)], 5 areas of each single gastric mucosa section were randomly selected for quantification. The number per square millimeter was counted and analyzed using Leica X image analysis software (Leica, Hamburg, Germany) and ImageJ software (National Institutes of Health, MD, USA).

Cells were fixed with 4% paraformaldehyde on ice for 15 min. Then cells were permeabilized in methanol in −20 °C for 30 min. Then cells were washed in blocking buffer (2% BSA in PBS) for 15 min, 3 times and stained in primary antibody at 4 °C overnight. On the next day, cells were washed (blocking buffer for 15 min, 3 times), followed by secondary antibody incubation for 1 h at room temperature in the dark. Then cells were washed and stained in VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Cat#H-1200) for 5 min and washed in a blocking buffer. Immunofluorescent images were obtained with a Leica TCS SP8 Inverted Fluorescence Microscope (Leica Microsystems) and analyzed with Leica X image analysis software (Leica) and Image J.^{72 (link)}