Rat anti mouse pecam 1 cd31

All experiments involving animals were authorized by the National Animal Experimental Board in Finland (Helsinki, Finland), under the licenses ESAVI/6285/04.10.07/2014 and ESAVI/403/2019.
Intracranial implantation of U87MG-GFP (n = 10) or BT-12 (n = 10) cells was performed as previously described [15 (link)]. Briefly, 8-week-old female NMRI:Rj nude mice were implanted with 10⁵ cells in 10 µL in the right striatum. After 20 days of tumor growth, 100 µg of PEI-MSNs in PBS were injected in the caudal vein (100 µL, n = 5) or intranasally (3 dosages of 5 µL given every two hours, n = 5). After 8 h, animals were euthanized, and brains were snap-frozen in −50 °C isopentane (Honeywell, Charlotte, NC, USA). Brain cryosections (9 µm) were cut using a cryotome (Thermo Fisher Scientific, Waltham, MA, USA), collected on Superfrost Ultra slides (Thermo Fisher Scientific, Waltham, MA, USA), and fixed in an ice-cold 4% PFA bath. Brain microvessels were stained overnight using a rat anti-mouse PECAM-1/CD31 (1:400, 553370, BD Pharmingen, Franklin Lakes, NJ, USA). Cell nuclei were counterstained with DAPI (1 µg/mL, Sigma), where after the samples were mounted with Mowiol 4-88 and imaged on a Zeiss LMS880 confocal microscope. The in vivo protocol was conducted twice.

Cryosections were blocked with 1% skim milk in PBS with 0.5% Tween-20 at RT for 15 min and incubated with primary monoclonal antibody (rat anti-mouse PECAM-1 [CD31; BD Pharmingen, San Diego, CA, USA; 1:400] or mouse monoclonal anti-QH-1 [Developmental Studies Hybridoma Bank; 1:100]) at 4 °C overnight. The sections were washed in PBS four times at RT and incubated with Alexa Fluor–conjugated secondary antibodies (Abcam, 1:400) for 1 hr. The sections were washed in PBS five times and mounted with Aqua-Poly/Mount (Cosmo Bio Co., Ltd., Tokyo, Japan). Nuclei were visualized with DAPI (Molecular Probes, Eugene, OR, USA). Fluorescence signals were visualized under a computer-assisted confocal microscope (Nikon D-Eclipse C1), and images were processed using NIS Elements software (Nikon, Tokyo, Japan). The same protocol was followed for whole-mount hearts and embryos, except that they were incubated with secondary antibodies overnight. Some sections were incubated with biotin-conjugated secondary antibodies and visualized using the Vectastain ABC System (Vector Laboratories, Burlingame, CA, USA).