Lative to baseline.Vascular and brain tissue collectionMice were killed by
Lative to baseline.Vascular and brain tissue collectionMice were killed by cervical dislocation and middle cerebral artery (MCA) segments Luteolin 7-O-��-D-glucoside biological activity immediately PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25681438 tested in vascular reactivity studies. For immunohistochemistry (IHC), western blot (WB) and ELISA studies, mice were exsanguinated by intracardiac perfusion of sterile 0.9 NaCl under deep sodium pentobarbital anesthesia. Vessels of the circle of Willis and their branches free of pial membrane along with cortex and hippocampus of one hemibrain were collected, snap-frozen on dry ice and stored (-80 ). The other hemibrain was fixed by overnight immersion in 4 paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (pH 7.4), cryoprotected, frozen in isopentane (-45 ) and stored (-80 ) until cutting into 25 m-thick free-floating coronal sections using a freezing microtome.secondary antibodies (1:300) for the detection of activated astrocytes, microglia, diffuse and dense-core A plaques, or LRP-1, respectively. Sections were observed under a Leitz Aristoplan microscope using bright field or an FITC filter and epifluorescence (Leica, Montr l, QC, Canada) and digital pictures were acquired with a digital camera (Coolpix 4500; Nikon, Tokyo, Japan). For double immunofluorescence, sections were simultaneously incubated with a rabbit anti-B1R antibody and either a guinea pig anti-GFAP, rat anti-CD11b, or mouse anti-A1?6 antibody, followed by donkey anti-rabbit Cy3- and speciesspecific Cy2-conjugated IgGs. Triple immunolabeling was also performed by co-incubating sections with rabbit antiB1R (with Cy5 secondary), rat anti-CD11b (Cy2) and mouse anti-A1?6 (Cy3) antibodies. Sections were observed and images acquired under a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss Ltd., Toronto, ON, Canada) equipped with appropriate filters.Staining quantification Vascular reactivityIn order to assess the impact of B1R blockade on the reactivity of cerebral vessels, isolated, pressurized and submaximally precontracted (5-HT, 2.10-7 M) MCA segments (diameter 40 to 70 m) from WT and APP mice were tested for dilatation to ACh (10-10 to 10-5 M) and BK (10-10 to 10-5 M) using online videomicroscopy PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 [31]. Percentage changes in vessel diameter from pre-constricted tone were plotted as a function of agonist concentration. The maximal response (EAmax) and the concentration eliciting half of EAmax (EC50 value, or pD2 = -[log EC50]), were generated by the GraphPad Prism software (version 4, San Diego, CA, USA) and used to evaluate agonist efficacy and potency, respectively.Immuno- and histochemical stainingSections were pretreated with 3 H2O2 (20 min) and incubated overnight at room temperature (RT) with either rabbit anti-B1R (1:1500) or -Egr-1 (1:250) antibodies diluted in a blocking buffer, followed by biotinylated anti-rabbit IgGs and the ABC kit; labeling was revealed with 0.05 DAB (B1R) or SG (Egr-1). To study basal Egr-1 expression levels and avoid task-induced changes [32], Egr-1 immunohistochemistry was done on animals sacrificed three days post-water maze. For detection of dense core amyloid plaques, sections from APP mice were stained with 1 thioflavin-S (8 min). Sections from all groups were incubated with rabbit anti-GFAP (1:400), -Iba1 (1:300), mouse anti-A1?6 (1:1000), or goat anti-LRP-1 (1:250), followed by species-specific Cy2- (GFAP and Iba1) or Cy3- (LRP-1) conjugatedDigital images (two to three sections/mouse, n = 4 to 6 mice) taken under the same conditions were analyzed with MetaMorp.