Nbp2 24683

Immunohistochemical staining was carried out following a previously published method 35 (link). Sliced colon tissue (4 μm) was embedded in paraffin and dewaxed and hydrated with dimethyl-benzene and alcohol, respectively, by the avidin-biotin complex (ABC) Peroxidase Staining Kit (Thermo Scientific, WA, USA). The samples were permeabilized with Triton-X 100 for 15 mins. Next, the sections were placed in citrate buffer (pH = 6.0), microwaved for antigen retrieval, and blocked in 3% H₂O₂ for 30 mins and donkey serum for 1 h. Finally, the sections were incubated at 4 °C overnight with the following corresponding primary antibodies: SP (abs137008, 1:100, Absin, Shanghai, China), proliferating cell nuclear antigen (PCNA, 60097-1, 1:500, Proteintech, Wuhan, China), zona occludens 1 (ZO-1, ab59760, 1:100, Abcam, Cambridge, MA, USA), STING (NBP2-24683, 1:200, NOVUS, Missouri, USA) and NK1R (DF7438, 1:200, Affinity, Michigan, USA). The sections were incubated with biotin-labeled horseradish peroxidase. After that, the tissues were stained using 3,3′-diamino-benzidine-tetrahydrochloride (DAB). Images were obtained by a microscope (Thermo Fisher Scientific, WA, USA).

Tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany). STING and cGAS immunohistochemistry was performed on paraffin-embedded tissue sections, using a polyclonal antibody against mouse/human STING raised in rabbit (NBP2-24683, 1:250, Novus Biologicals, Denver, CO, USA or 13657, 1:250, Cell Signaling Technology, Danvers, MA, USA) or against mouse/human cGAS raised in rabbit (201708-T10, 1:125, Sino Biological, Eschborn, Germany). For fluorescent microscopy of STING and cGAS, murine or human preadipocytes were grown on optical transparent glass-bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany) or glass coverslips and labeled with the same antibodies used for immunohistochemistry, and then visualized with AF488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Histology images were adjusted to equal white balance after acquisition. Flow cytometry analysis was used to detect STING⁺, cGAS⁺ macrophages and adipocytes, as described in [24 (link)]. Flow repository identifier of FACS data is FR-FCM-Z236.

On day 8 after GCV injection, the mice were completely anesthetized with isoflurane and then perfused with sterile saline throughout the body, followed by fixation with 4% paraformaldehyde. After fixation, the segment where the mouse colon was located was taken and the same fixative was left overnight. After sucrose gradient dehydration, the tissues were embedded with OCT tissue embedding agent and frozen at −80 °C in the refrigerator. The embedded tissues were sectioned (20 μm thickness) and processed on a cryostat microtome (CM 1950; Leica Microsystems, Wetzlar, Germany). Sections were incubated with 5% goat serum and incubated overnight at 4°C with primary anti F4/80 antibody (Abcam Cat#ab6640, RRID: AB_1140040) and anti-STING antibody (Novus Cat# NBP2-24683, RRID: AB_2868483). Then after washing away the first antibody, sections were incubated with FITC and Cy3 conjugated secondary antibodies (goat anti-rabbit Alex Flour 555 pAb Cell Signaling Technology Cat#4413, RRID: AB_10694110) (goat anti-rabbit Alex Flour 488 pAb Abcam Cat#ab150157, RRID: AB_2722511) for 1 h at room temperature (Ding et al., 2022 (link)). Immunostained sections were examined under a ZEISS fluorescence microscope (Carl Zeiss OPMI Pentero, Germany), images were taken and the sections were examined with NIH ImageJ software (NIH, Bethesda, MD) for analysis.