Aqueous mounting medium

Cells were centrifuged at 800×g onto glass slides, and coverslips were mounted with aqueous mounting medium (Dako) containing DAPI (Sigma–Aldrich). Fluorescence signals were analyzed using a Zeiss LSM 700 laser-scanning confocal microscope. LC3 puncta were quantified in cells as described [33 (link)]. The average number of LC3 puncta per cell in each treatment group was estimated by manually counting puncta in 20 randomly selected cells.

Tissue was fixed in 4% PFA (Biognost, cat. no. FNB4) for up to 48 h, dissected coronally in three blocks, embedded in paraffin (Merck, cat. no. 107300) and sectioned on a microtome (Leica, SM2000R) at 20 μm thick sections. Prior to immunohistochemistry, a standard process of deparaffinization was performed in a series of xylol and alcohol. After four washes in 1x PBS, blocking solution containing 1% BSA and 0.5% Triton X-100 in PBS was applied on sections for 1 h. Blocking solution was replaced with primary antibodies (mouse anti-Elavl4, 1:1000; rabbit anti-Pax6, mouse anti-Celf1, 1:200) diluted in blocking solutions and kept overnight at 4 °C. After incubation, sections were 3× washed in 1× PBS and appropriate secondary antibodies (donkey Alexa Fluor 546 (cat. no. A10036) and Alexa Fluor 488 (cat. no. A21206), diluted 1:1000, Thermo Fisher Scientific) were applied for 2 h. Following 3× washes in 1× PBS, DAPI (Sigma Aldrich, St. Louis, USA) was applied for 1 min according to manufacturer instructions. Sections were covered in Aqueous Mounting Medium (DAKO, Carpinteria, CA, USA).

BrdU immunohistochemistry was performed as described^{26 (link)}. Paraffin sections were dewaxed and underwent antigen retrieval, followed by denaturation with 0.1 N HCl for 45 min. The sections were then quenched with 3% hydrogen peroxide for 10 min, blocked with 5% BSA/PBS for 1 h, and incubated with sheep anti-BrdU antibody (1:500 dilution; Abcam) or isotype control at 4 °C overnight. On the next day, the sections were incubated with donkey anti-sheep biotinylated secondary antibodies (1:400 dilution; Abcam) for 1 h and then with the Vectastain ABC reagent (Vector Laboratories) for 30 min. BrdU positive staining was revealed by DAB solution (Dako) under a Zeiss Axioskop II microscope (Carl Zeiss). The sections were counterstained with the Mayer’s hematoxylin, dehydrated, and mounted using aqueous mounting medium (Dako). Images were acquired using a Photometrics CoolSNAP digital camera (Roper Scientific).

Immunohistochemical staining for RAD51, and 53BP1 was performed using the DakoEnVisionTM kit (Dako, Glostrup, Denmark). After dewaxing in xylene and rehydration in graded alcohols, tissue sections were boiled in citrate buffer. Sections were incubated with primary antibody. And staining was completed by incubation with 3,3-diaminobenzidine (DAB) and substrate chromogen, which results in brown-colored precipitate at the antigen site. Finally, sections were counterstained with haematoxylin and mounted in Aqueous Mounting Medium (Dako, Glostrup, Denmark).

The equivalent of 10-µg protein of PKH67-stained EVs was incubated with a primary culture of embryonic thyroid cells. After 3 h, cells were rinsed extensively with PBS to remove remaining particles, fixed with 4% formaldehyde pH 7.4 for 15 min, and then washed again extensively. Cells were further permeabilised with 0.05% saponin pH 7.4 for 15 min and incubated for 30 min in blocking solution (1% BSA; 0.1% lysine; 0.01% saponin in PBS). Permeabilised cells were then incubated with anti-CD63 (1:150, Biolegend) primary antibody, diluted in blocking solution for 1 h, washed with blocking solution, incubated with secondary antibody coupled with Alexa-568 (1:1,000, Invitrogen) together with Hoechst 33258 as nuclear dye (1:1,000, Sigma) in blocking solution for 1 h. After final wash in blocking solution, cells were rinsed three times with PBS, post-fixed in 4% formaldehyde for 5 min and mounted in aqueous mounting medium (Dako). Preparations were observed with a Zeiss Cell Observer Spinning Disk microscope (objective 100×, oil immersion).