Vectashield medium

The desired brain sections containing the SFO and OVLT were initially washed free‐floating in PBS. Next, brain tissue was permeabilized with 0.1% Triton X100 (Sigma) for 20 minutes at room temperature (RT), following further washing in PBS. Slices were then blocked in 5% normal donkey serum (NDS; Sigma) and 0.05% Triton X100 at RT for 30 minutes. A 48 hours incubation at 4°C in a PBS solution containing 0.05% Triton X100, 0.5% NDS and the primary antibodies rabbit anti‐GFP (1:1000, Abcam), and chicken anti‐vimentin (1:4000, Abcam). Following this incubation, slices were washed in PBS and transferred to PBS solution of secondary antibodies for 24 hours at 4°C; donkey anti‐rabbit Cy5 (1:800, Jackson ImmunoResearch) and donkey anti‐chicken Alexa 488 (1:800, Abcam). The secondary antibodies were then washed off in PBS and the slices were mounted on gelatine‐coated glass slides using VectaShield medium (Vector Laboratories, USA). Slices were then imaged on a confocal microscope (Leica SP5 upright) under the 20x objective. Z‐stacks were acquired in 3 µm steps and viewed as maximal intensity projections.

vMSCs were seeded on cover glasses at the density of 1.5 × 10⁴ cells/glass for 24 h. Then, the medium was changed with a fresh one containing 50 ng/ml of vascular endothelial growth factor (VEGF) for 7 days at 37° and 5% CO₂, followed by morphine sulphate treatment for other 7 days at 37 °C and 5% CO₂. At the end of the treatment, the samples were fixed in 4% paraformaldehyde in PBS for 30 min at 4 °C, followed by a permealization step in 0.1% Triton X-100 for 5 min at 4 °C. After some washes in PBS, the samples were covered with 2.5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS (blocking reagent), for 30 min at room temperature (RT), followed by incubation in mouse anti-human CD31 antibody (Origene, Thermo Fisher Scientific, Monza, Italy) diluted 1:100 in blocking reagent, overnight at 4 °C. Then, the samples were washed in PBS and incubated for 1 h and 30 min at 37 °C with secondary antibody anti-mouse IgG–Cy3 conjugated (Sigma-Aldrich, St. Louis, MO, USA), diluted 1:2000 in PBS. Samples were rinsed with PBS, counterstained with DAPI and mounted in vectashield medium (Vector Laboratories, Inc., Burlingame, CA, USA). The fluorescence microscopy Eclipse E800 Nikon (Nikon, Tokyo, Japan) was utilized to observe the samples.

The immunofluorescence method used here was described in our previous research study^{42 (link)}. Thirty micrometer-thick cryostat sections were incubated with primary antibodies against rabbit anti-tyrosine hydroxylase (TH, 1:1000, Millipore, Temecula, CA, USA) in 1% BSA overnight at 4 °C. This is a specific marker of DA neurons. After the sections were washed with 0.01 M phosphate buffer, they were probed with the appropriate Cy3- and Alexa 488-conjugated secondary antibodies (1:500 for 4 h at room temperature (RT); Jackson ImmunoResearch, West Grove, PA, USA). Finally, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology) and mounted in Vectashield medium (Vector Laboratories) before they were photographed using a Zeiss confocal microscope (Zeiss, LSM780).

Retinal neovascularization was induced by the use of a well-established murine model of oxygen-induced retinopathy [20 (link)]. Neonatal mouse (C57BL/6) pups at postnatal day 7 (P7) with their nursing mothers were maintained for 5 days in 75% oxygen and then returned to room air (relative hypoxia) to produce retinal neovascularization at P12. PBS, scramble, KC21 peptides (25 μg), or Bev (10 µg) were then administered by intravitreal injection into mouse eyes at P12. The animals were sacrificed and the mouse eyes were enucleated at P17. Mouse eye cups were fixed in 4% paraformaldehyde for 2 h. The retinas were carefully separated from eye cups and then incubated with fluorescein-labeled isolectin-B4 (Life technologies) at 4 °C overnight. Samples were mounted with Vectashield medium (Vector Laboratories), and the isolectin labeling was examined by using the × 20 objective of a Leica TCS SP5 confocal microscope. Fluorescence volume measurements were recorded by creating image stacks of optical slices within lesions with QWIN software.

Human fibroblasts were arrested in metaphase with 1 μg/ml colcemid (2 h, 37°C) (KaryoMAX colcemid; Life Technologies), trypsinized, resuspended in serum-containing medium, centrifuged (1,500 rpm, 5 min), resuspended in 5 ml of 75 mM KCl, and incubated at RT (10 min). Seven drops of fresh 3:1 methanol-acetic acid fixative were then added, and cells were centrifuged (1,500 rpm, 5 min). After removing all but 100 μl of the fixative, cells were resuspended in 4.5 ml of fresh fixative added dropwise while vortexing at low speed and then incubated in fixative overnight (4°C), centrifuged (1,500 rpm, 5 min), resuspended in 1 ml of fresh fixative, pelleted in a microcentrifuge at top speed, washed with fixative, again pelleted in a microcentrifuge at top speed for 2 min, and resuspended in 250 μl fixative. One hundred microliters of suspension was dropped onto ethanol-precleaned microscope slides and dried in a fume hood (1 h). The slides were washed in ultrapure water and mounted in DAPI (4′,6′-diamidino-2-phenylindole)-containing Vectashield medium (Vector Laboratories) prior to deconvolution microscopy.