Anti guinea pig cy3

Immunohistochemistry was performed as described previously[21 (link)] on 10 μm frozen sections of cold phosphate-buffered saline perfused brains taken from mice subject to normoxia (control) or hypoxia for 4, 7, and 14 days. Each slide contained mouse brains representing the four different time-points of hypoxia, to ensure consistent antibody incubation times across different time-points. The following rat monoclonal antibodies were obtained from BD Pharmingen (La Jolla, CA, USA): anti-CD31 (clone MEC13.3), anti-β4 integrin (clone 346-11A), and anti-CD151 (clone 455807). Other primary antibodies used included mouse anti-α-SMA-Cy3 conjugate (Sigma, clone 1A4), hamster anti-CD31 (clone 2H8, Abcam, Cambridge, MA, USA), rabbit anti-CD151 (Creative Diagnostics, Shirley, NY, USA), and guinea pig polyclonal anti-plectin (Progen, Heidelberg, Germany). Secondary antibodies used included anti-rat-Cy3, anti-rabbit-Cy3, and anti-guinea pig-Cy3 (Jackson ImmunoResearch, West Grove, PA, USA), anti-Armenian Hamster-Dy-Light 594 (BioLegend, San Diego, CA, USA), and anti-rat Alexa Fluor 488 and anti-guinea pig Alexa Fluor 488 (Invitrogen Corporation, Carlsbad, CA, USA).

Intact brains from postnatal day 3 (PD3), postnatal day 7 (PD7), 2 weeks, and 7 weeks old male mice were isolated from WT and nNOS^−/− mice and immediately placed in 4% paraformaldehyde (PFA) for 48 h. Brains were subsequently transferred to a 30% sucrose solution for at least 48 h and then sagittally sectioned at a thickness of 80 μm using a vibratome, placed in a cryoprotectant solution, and stored in −20°C until stained. Slices were first washed with PBS, then permeabilized using a 0.25% Triton‐X solution for 5 min. Free‐floating slices were then blocked with a 10% normal donkey serum (NDS) solution for 1 h and incubated with the following primary antibodies overnight in 4°C: 1:500 goat anti‐calbindin (CalB) (Santa Cruz Biotechnology, Dallas, TX); 1:500 guinea pig anti‐GLAST (Synaptic Systems, Goettingen, Germany). Slices were washed and then incubated with the appropriate secondary antibodies for 2 h: 1:1000 anti‐goat AlexaFluor 488, or 1:500 anti‐guinea pig Cy3 (Jackson Immunoresearch, Burlington, ON). After incubation with the nuclear stain DAPI, slices were mounted on cover glass using Fluoromount‐G (Electron Microscopy Solutions, Hatfield, PA) and images were taken using the Olympus FV1000 confocal microscope at ×60 magnification using an oil‐immersion objective.