E ab 1003

Exosomal proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE kits, Boster Biological Technology Co., Ltd. Wuhan of China) and transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA) by wet electro-transfer. Membranes were blocked in 5% non-fatty milk for 1 h. Then primary antibodies were added for overnight incubation at 4 ℃ (anti-CD9: SBI, EXOAB-CD9A-1, 1:1000; anti-CD81: Immunoway, YT5394, 1:1000). After washing by 0.1% PBST (10 min × 3 times), the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Elabscience, E-AB-1003, 1:4000) for 1 h at room temperature. After washing by 0.1% PBST (10 min × 3 times), enhanced chemiluminescence (ECL) detection kit (Boster Biological Technology Co., Ltd. Wuhan of China) was used to detect protein bands by Tanon-5200 automatic chemiluminescence imaging analysis system (Tanon Science & Technology Co., Ltd. Shanghai of China).

The iWAT, eWAT, pWAT, liver, and pancreas were fixed in 10% formalin buffer for 48 h, embedded in paraffin, and sliced into 5 μm thick sections. For Oil Red O staining, the sections of liver were stained with 3.7 mM Oil Red O dissolved and then counterstained with hematoxylin as in a previous study [33 (link)]. For hematoxylin and eosin (H&E) staining, the sections were stained with hematoxylin for 5 min and eosin for 3 min at 25 °C as in our previous study [23 (link)]. For immunohistochemistry staining, the sections of pancreas were blocked with 5% bovine serum albumin (Gen-view Scientific, Galveston, TX, USA), then incubated with anti-insulin antibody (#ab181547, 1:2000 dilution) (Abcam, Cambridge, MA, USA) at 4 °C overnight. The sections were then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (#E-AB-1003) (Elabscience, Wuhan, China) at 4 °C for 2 h. Color development was performed using a DAB Kit (#34065, Thermo Fisher, Carlsbad, CA, USA), and hematoxylin was used for counter staining.
The stained slides were observed under a light microscope (Olympus, Tokyo, Japan) and photographed.

The total proteins from each sample were separated by electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (IPVH00010, Merck Millipore, USA). After being blocked with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4° C overnight (Supplementary Table 2). Then, the membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, E-AB-1003, Elabscience, China) for 1 h at room temperature. The proteins on the membranes were visualized with Immobilon Western HRP Substrate (WBKLS0500, Merck Millipore) and scanned on a SuperSignal chemiluminescent detection system (BioRad, USA).

The colorectum, heart, liver, spleen, kidney, and lung specimens collected from Apc^Min/+ mice were fixed in 4% paraformaldehyde for 48 h. Samples were embedded in paraffin, sliced into 5 μm sections, and stained with hematoxylin and eosin (H&E), as described in a previous study (Elsherbiny et al., 2017 (link)).
Other sections of the colorectum (especially the colorectal tumor portion) and spleen were blocked with 5% bovine serum albumin (Gen-view Scientific, Galveston, TX, United States ) for 4 h and incubated with primary antibodies including CD4 (25229S, 1:200 dilution) and CD8 (98941S, 1:800 dilution) (Cell Signaling Technology, Danvers, MA, United States ), IFN-γ (PA5-95560, 1:1000 dilution) and IL-4 (PA5-25165, 1:50 dilution) (Invitrogen, Carlsbad, CA, United States ) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (E-AB-1003 or E-AB-1001) (Elabscience, Wuhan, China) at 4°C for 2 h. Color development was performed using the Metal Enhan DAB Substrate Kit (34,065, Thermofisher, Carlsbad, CA, United States ), and hematoxylin was used for counterstaining. All the slides were observed under a light microscope (Olympus Corporation, Tokyo, Japan). ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify the pixel density for the semi-quantitative densitometric analysis of protein expressions.

Equal amounts of protein were electrophoresed on 10% SDS-PAGE gels and transferred to PVDF membranes for immunoblotting. The membranes were blocked with a 5% non-fat milk dilution in TBST for 1 hour at RT and incubated with dilutions of primary antibody, which is Orai2 (20592-1-AP, Protein tech, dilution ratio of 1:1,000) or GAPDH (AF7021, Affinity, dilution ratio of 1:1,000) kept overnight at 4°C. After incubation with HRP-conjugated goat anti-rabbit IgG (E-AB-1003, Elabscience, dilution ratio of 1:5,000), chemiluminescence detection was performed by using the ECL plus Western blotting detection reagent (GE Health, Little Chalfont, United Kingdom), and immunoblots were quantified by densitometry using a chemiluminescence gel imaging analysis system (P and Q, Shanghai, China).