Bx 63 upright fluorescent microscope

Organoids were fixed for histology on days 1 and 10 of culture in 4% paraformaldehyde for 4 hours. Organoids were processed, paraffin embedded, and sectioned at 5-μm intervals for staining. Organoid sections were stained on glass microscope slides with hematoxylin and eosin (H&E).
Additional staining was performed with immunohistochemistry (IHC) to characterize programmed death ligand-1 (PD-L1), cluster of differentiation 8 (CD-8), cytokeratin 20 (CK-20), and granzyme B biomarker expression. Unstained slides underwent antigen retrieval in a pH 6 citrate buffer solution prior to blocking with Dako Protein Block for 30 minutes. Fluorescent IHC was performed by applying primary antibodies PD-L1 (ab205921, abcam, rabbit), CD-8 (ab4055, abcam, rabbit), CK-20 (MA5–13263, Invitrogen, mouse), and granzyme B (ab4059, abcam, rabbit), and cleaved caspase 3 (9661S, Cell Signaling Technologies, rabbit) to slides in ratios of 1:500, 1:200, 1:200, 1:100, 1:400 in Dako Antibody Diluent, respectively. After incubation for 1 hour, appropriate species reactive secondary Alexa Fluor 488 or Alexa Fluor 594 antibodies (Biotium, Fremont, CA) were applied to samples for 1 hour at a 1:1000 dilution. Sections were then incubated with DAPI for 5 minutes prior to finalization with coverslipping. An Olympus BX-63 upright fluorescent microscope (Olympus, Tokyo, Japan) was used to image the sections.

DYN^tdTOM mice were sacrificed and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The lumbar enlargement of the spinal cord was removed and placed in 30% sucrose (in 0.1M PB) overnight. The tissue was frozen and 40 μm sections were cut using a cryostat (Leica; Wetzlar, Germany). Free-floating sections were washed in phosphate-buffered saline (PBS) and placed in a blocking buffer containing 10% normal donkey serum and 0.3% Triton X-100. The sections were then incubated in a primary antibody against the transcription factor Pax2 (1:500 dilution; ThermoFisher #716000; RRID: AB_2533990) overnight at 4°C. Pax2 was used as a marker of inhibitory neurons since it is continuously expressed throughout development and is present in both GABAergic and glycinergic neurons [39 (link); 52 (link); 62 (link)]. Sections were washed again in PBS and incubated in a species-specific secondary antibody conjugated to AlexaFluor 488 (1:500 dilution; ThermoFisher #A-11008; RRID: AB_143165) for one hour at room temperature.
The spinal cord dorsal horn of each section was imaged on a BX63 upright fluorescent microscope (Olympus; Center Valley, PA) at 20X or 40X, and z-stack images were taken with a separation of 1 μm. The total number of both tdTOM- and tdTOM/Pax2-expressing cells in a section were counted using CellSens Dimension Desktop software (Olympus).

EV-containing SEC and DMC fractions were incubated with CM-DiI (Thermo Fisher Scientific) for 30 min at room temperature. EV-free samples were subjected to the same labeling processes. Dye aggregates were removed by Millex-GV syringe filter (0.22 μm pore size, Millipore Sigma). Filtered EVs were captured on a glass slide. Following 30 min incubation at room temperature, the slide was washed with PBST (PBST buffer containing 0.001% Tween 20). After incubation with fixation buffer (4% formaldehyde) and blocking buffer (Superblock, Thermo Fisher Scientific), EV samples were incubated with anti-human CD63 antibody (Ancell) for 90 min at room temperature. After washing, Alexa Fluor 488-labeled secondary antibody was introduced and incubated for 30 min at room temperature. After final wash steps, fluorescence images were taken with BX-63 upright fluorescent microscope (Olympus) with 40× objective. Acquisition settings (i.e., objective, exposure time, camera setting, illumination) were kept constant for all images.