Streptavidin cy5

Following recording, slices containing neurobiotin-filled neurons were fixed overnight in Lana’s fix. Slices were subsequently washed four times in 0.01 M PBS, then placed in 0.5 μl streptavidin Cy5 (Thermo Scientific, USA) in 500 μl 0.1 M PB containing 0.3% Triton X-100 overnight. For TH-staining, slices were incubated in a 1:1000 Anti-Tyrosine Hydroxilase antibody (AB152, lot 3114503, Millipore Sigma, USA). Slices were washed again before mounting on Superfrost Plus microscope slides (Thermo Scientific, USA) using Fluoromount Aqueous mounting medium (Sigma, USA). Mounted tissue images were subsequently acquired using the Zen 2 black edition software (Carl Zeiss AG, Germany).

Cells were fixed with 4% paraformaldehyde (Carlo Erba, Milan, Italy), permeabilized by 0.5% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) and incubated for 1 h at 4 °C with the following antibodies, at a final concentration of 0.1 mg/mL: anti-AMOT1 (Santa Cruz Biotechnology, Inc. Dallas, TX, USA; sc-166924), anti-YAP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or anti-p-YAP (Cell Signaling Technology). For F-actin detection, cells were stained with Biotin-XX Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA; #B7474). After washings, cells were incubated for 30 min at 37 °C with a secondary antibody conjugated with Cy5 (Abcam) or Streptavidin-Cy5 (Thermo Fisher Scientific, Waltham, MA, USA). Cell samples were washed twice in PBS and immediately acquired by a cytometer.
For flow cytometry studies, samples were acquired with a FACScalibur cytometer (BD Biosciences Inc., San Diego, CA, USA) equipped with a 488 nm argon laser and with a 635 nm red diode laser. At least 20,000 events were acquired, recorded and analyzed using CellQuest software (BD Biosciences, San Diego, CA, USA). The expression level of the analyzed proteins by flow cytometry was reported as median fluorescence.

Cells were fixed for 15 min at room temperature (RT) in paraformaldehyde (PFA, 4% wt/vol, Sigma-Aldrich) and sucrose (14% wt/vol, Sigma) solution prepared in PBS (1×). After washes in PBS, cells were permeabilized with Triton (0.25% vol/vol, Sigma-Aldrich) diluted in PBS. Cells were washed again in PBS and incubated for 1 h at RT in Triton (0.1% vol/vol, Sigma-Aldrich) and goat serum (GS, 10% vol/vol, Invitrogen) in PBS to block nonspecific staining. Subsequently, neurons were incubated for 1 h with a primary antibody mix consisting of guinea pig antibodies against GABA_AR α2 subunit (1:2000, provided by J.M. Fritschy, University of Zurich) and rabbit anti-VGAT (1:400, provided by B. Gasnier, University Paris Descartes, Paris) in PBS supplemented with GS (10% vol/vol, Invitrogen) and Triton (0.1% vol/vol, Sigma-Aldrich). After washes, cells were incubated for 60 min at RT with a secondary antibody mix containing biotinylated F(ab′)2 anti-guinea pig (1:300, Jackson Immunoresearch) and AMCA350-conjugated goat anti-rabbit (1:100, Jackson Laboratory) in PBS-GS-Triton blocking solution, washed, incubated for another 45 min with streptavidin-CY5 (1:300, Thermo Fisher Scientific), and finally mounted on glass slides using Mowiol 4-88 (48 mg/ml, Sigma-Aldrich). Sets of neurons compared for quantification were labeled simultaneously.

The HuProt microarray(CDI Laboratories, Inc.) was composed of 20,240 human full-length proteins with N-terminal glutathione S-transferase (GST) tags. The HuProt microarray assay was performed by Wayen biotechnologies (Shanghai), Inc. according to the following procedure. Human Proteome microarrays (HuProtTM 20 K) were blocked with blocking buffer (1% BSA in 0.1% Tween 20, TBST) for 1 h at room temperature with gentle agitation. ANGPTL3 protein (ab176028, abcam, USA) was labeled with biotin by the Antibody Array Assay Kit (Full moon Biosystems, Sunnyvale, CA), and then diluted to 0.01 mg/ml in blocking buffer and incubated on the blocked proteome microarray at room temperature for 1 h. The microarrays were washed three times for 5 min each time with TBST, incubated with streptavidin-Cy5 at 1:1000 dilution (Thermo Fisher Scientific, USA) for 1 h at room temperature and underwent three more 5-min washes. The microarrays were spun dry at 1500 rpm for 3 min and subjected to scanning with a Genepix 4000B (Axon Instruments, Sunnyvale, CA) in order for results to be visualised and recorded. A GenePix Pro 6.0 was used for data analysis.

AlexaFluor488 secondary anti-mouse antibody, goat anti-human biotin conjugate antibody and streptavidin-Cy5 were purchased from Life Technologies and Abcam. Purified EVs were fixed by adapting standard methods^{72 (link)} in the following way: Coverslips (Menzel-Glaeser) washed with water (2×), ethanol (1×), water (2×) and methanol (1×) were carefully flame dried. Volumes (10–40 μl) of EVs resuspended in PBS were applied to the coverslips and left to air dry before dipping them in cold methanol. Samples were blocked with filtered 3% BSA (Sigma-Aldrich) in PBS for 1 h. Primary antibodies in 3% BSA PBS were incubated for 1 h and washed five times for 5 min with 3% BSA PBS. Secondary antibodies, also in 3% BSA PBS, were incubated for 45 min and then washed five times for 5 min with 3% BSA PBS. Prior to imaging, coverslips were air dried and 5 μl of mounting medium with DAPI (Vectashield, Vector Laboratories) was applied before sealing them on microscope slides with nail polish. Sorcin-SR1was obtained commercially (Abcam).