Apc conjugated f4 80 antibody

The resected mice colon tissues were washed with cold PBS followed by immediate fixation in 4% PFA then stored at 4 °C overnight. For histopathological analysis, paraffin-embedded tissue sample were sectioned (4 µm) and stained with hematoxylin and eosin (H and E). All images of sections were obtained using a BZ-X710 All-in-one inverted fluorescence microscope (Keyence, Osaka, Japan) with 10× and 20× objective lenses. The images were analyzed using BZ-X analyzer software (Keyence) and ImageJ software (NIH, Bethesda, MD, USA). For the immunohistology of F4/80 positive cells in colon tissues, frozen OCT-embedded tissue samples were sectioned (8 µm) at −20 °C then post-fixation with 4% PFA for 10 min. Permeabilization was performed with 0.5% triton x-100 in PBS with 15 min. The sections were blocked by 10% goat serum (Nichirei Bioscience, Tokyo, Japan) for 30 min. Immunostaining of F4/80 was performed with APC conjugated F4/80 antibody (123115, BioLegend, San Diego, CA, USA) at 4 °C, overnight. Sections were mounted with Prolong gold antifade mountant with DAPI (Thermo Scientific, Waltham, MA, USA). The images were captured with LSM-780 confocal laser microscopy (Carl Zeiss, Jena, Germany). The excitation wavelength was set with 633 nm laser for APC detection and 405 nm laser for DAPI. The images were analyzed using Zen software (Carl Zeiss).

The 10-day-old ICR mice were i.g. inoculated with 10⁵ TCID₅₀ CVA6 strains. At 1 dpi, 3 dpi, 5 dpi, and 7 dpi, mock- and CVA6-infected mice (n = 7–10) were euthanized. The brain and spleen cells were extracted and staining as described previously [31 (link),32 ]. The antibodies used in FACS analysis were purchased from Biolegend Inc., CA, USA: APC-conjugated CD3 antibody (#100312), AF700-conjugated B220 antibody (#103210), BV711-conjugated CD4 antibody (#100549), Biotin anti-CD8 antibody (#100704), FITC-conjugated CD44 antibody (#103006), PE-conjugated CD69 antibody (#104507), FITC-conjugated CD25 antibody (#101907), FITC-conjugated Ly-6c antibody (#128006), PE-conjugated CD62L antibody (#104407), PE-conjugated CD11c antibody (#117308), PerCP/Cy5.5-conjugated Ly-6G antibody (#127615), APC-conjugated F4/80 antibody (#123115), APC/Cy7-conjugated CD45 antibody (#103116), PerCP/Cy5.5-conjugated IgM antibody (#406512), PE/Cyanine7 Streptavidin (#405206), anti-CD16/32 antibody (#101330). LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific Co., Ltd, Waltham, USA) was used to distinguish dead cells from living ones. Flow cytometry measurement was performed using the BD LSRFortessa FACS Canto and the data was analyzed by the FlowJoX software.