Peroxidase labelled secondary antibody

Sections were dewaxed in xylene (Fisher Chemicals), rehydrated in graded alcohols (Fisher Chemicals), and washed in tap water. Antigen retrieval, hydrogen peroxidase blocking, serum block with NChS, and primary antibody incubation were performed as described for immunohistochemistry. The following day, sections were washed with TBS and incubated with peroxidase labelled secondary antibody (Santa Cruz Biotechnology) at 1:200 in NChS for 30 min, followed by washes in TBS and incubation with Tyramide signal amplification (TSA, Perkin Elmer, Waltham, MA, USA), at 1:50 for 10 min. Antigen retrieval was then performed by microwaving the sections at full power for 2.5 min in 0.01 M citrate buffer, followed by a 30 min cool-down period. The process from NChS block up to primary antibody detection was repeated twice more for two subsequent primary antibodies. Sections were counterstained with DAPI (4′, 6-diamidino-2-phenylindole, Sigma-Aldrich) by adding 1 µL/mL of TBS and incubating the sections for 10 min in the dark. Finally, sections were washed with TBS and mounted with PermaFluor (Life Technologies, Paisley, UK). Primary antibody details are described in Table 1.

EV sample was concentrated using Concentrator plus 5305 Vacuum Centrifuge (Eppendorf AG, Germany), and protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Scientific). Concentrated sEV preparations and lysate of HCT116 cells were lysed in Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific), heated for 5 min at 95 °C, and subjected to electrophoresis using 10% SDS-PAGE. Proteins were transferred to an Immobilon-P PVDF Membrane (Merck Millipore) and the excess protein binding sites on the membrane were saturated with 5% bovine serum albumin blocking buffer (1 × TBS, 0.1% Tween-20) for 1 h. The membrane was incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-CD81 (1:250, mouse, catalogue number sc166029), anti-CD63 (1:300, mouse, sc5275) from Santa Cruz Biotechnology, anti-Alix (1:20000, rabbit, ab186429) from Abcam, anti-TSG101 (1:200, mouse, 612696) from BD Biosciences, and anti-Calnexin (1:1000, rabbit, 2679) from Cell Signaling. After incubation, the membrane was washed three times with 5% TBS-Tween and then, incubated with peroxidase-labelled secondary antibody (Santa Cruz Biotechnology) for one hour. After three washes, immobilized proteins were detected utilizing Clarity Western ECL Substrate (Bio-Rad) and the UVITEC chemiluminescence imager (UVITEC Cambridge, UK).