Horse serum

Animals (n = 2 in each condition) were perfused with phosphate-buffered saline (PBS, pH 7.4) containing 4% (w/v) paraformaldehyde (PFA) and 0.2% (v/v) picric acid under general ketamine-medetomidine anesthesia. The brain was removed, incubated for 24 h in perfusion solution, and infiltrated with 30% (w/v) sucrose in PBS for at least 24 h. Coronal sections of 50 µm were cut on a Leica SM 2000R sliding microtome. The sections were rinsed three times in Tris-buffered saline (TBS, Sigma), three times in TBS containing 3% (w/v) Triton X-100 (TBS-T), and once for 1 h in TBS-T containing 20% (v/v) horse serum (Vector Labs) and then incubated for 48 h at 4°C in TBS-T containing 1% horse serum and combinations of the following primary antibodies: anti-GFP (chicken, 1∶500, AbCam), anti-parvalbumin (mouse, 1∶2,000, Swant), or anti-somatostatin (rabbit, 1∶500, Millipore). The sections were rinsed 4 times in TBS and stained in TBS-T containing 1% horse serum and Alexa488- and Alexa546-labeled secondary antibodies (Invitrogen). After four rinses in TBS, the slices were mounted in VectaShield (Vector Labs) and imaged on a Leica TCS SP5 confocal microscope.

HMVECs were grown on 8-well chamber slides to reach confluent monolayers. After being treated with different species of amyloid proteins (Aβ 20 μM, IAPP 20 μM, αS 10 μM and FapC 1.6 μM) for 30 min, cells were gently washed with Hank’s balanced salt solution (HBSS, Gibco, USA) and fixed by 4% paraformaldehyde (Sigma-Aldrich) for 15 min, followed by permeabilizing and blocking with 0.1% saponin (Sigma-Aldrich) and 5% horse serum (Sigma-Aldrich) in PBS/azide for 1 hour at room temperature. Thereafter, fixed cells were washed three times with PBS and incubated overnight at 4 °C with primary rabbit anti-VE-cadherin antibody (Abcam) at 1:400 dilution with 5% horse serum in PBS/azide. Then, the cells were washed with PBS and incubated with secondary donkey anti-rabbit Alexa Fluor 594 antibody (1:500, Abcam) and Phalloidin-iFluor 488 (1:1000, Abcam) in PBS/azide solution for 2 h at room temperature. After nuclei staining with Hoechst 33342 (Sigma-Aldrich, USA) at 1:2000 dilution for 5 min, the cells were imaged with Leica SP8 lightening confocal microscope (Leica, Germany) through an HC PL APO CS2×63/1.40 oil objective and semiquantitative analysis was performed using ImageJ.

Immunohistochemistry was performed as previously described (27 (link)). Tissue sections were incubated overnight at 4°C in an optimized blocking solution: 5% blotting-grade milk (Bio-Rad, Hercules, CA) and 5% horse serum (Vector Laboratories, Burlingame, CA, USA) diluted in double distilled H₂O. Rabbit monoclonal anti-P-Glycoprotein antibody (Abcam, Cat. ab170904) was diluted (1:1000 for all mouse tissues) in 5% milk and 5% horse serum as described above for overnight incubation at 4°C. The appropriate secondary antibody, anti-rabbit IgG made in horse (Cat. MP-7401, Vector Laboratories) was not diluted, and incubated at room temperature for 1 hour. Positive immunoreactivity was revealed via chromogenic detection with ImmPACT DAB Peroxidase (HRP) Substrate (SK-4105, Vector Laboratories) with incubating 3 minutes, then counterstained with hematoxylin (Gills Formula, Vector Laboratories) for five seconds and covered with a coverslip. Images were captured with a BX41 bright-field microscope (Olympus, Center Valley, PA, USA). Immunostaining intensity was quantified by using ImageJ software (NIH Public Domain) by a person blind to sample ID. Images were taken at 20x magnification from at least 5 randomly selected areas per skin specimen.