Alexa 647 conjugated anti rabbit igg

To quantify CAA burden and Aβ-associated fragmentation of smooth muscle cells in pial vessels [20 (link), 31 (link)], tangential brain sections were incubated with anti-Aβ (4G8, 1:1,000, mouse; Covance) and the smooth muscle marker anti-α-actin (1:300, rabbit, Abcam) for 48 h. After washing, sections were labeled with Alexa 488-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch) and Alexa 647-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch). Pial arterioles (n = 30–50/group) positive for Aβ and α-actin, ranging in diameter from 20 to 100 μm, were randomly imaged by confocal microscope (63x). Aβ accumulation around pial-penetrating arteries was assessed by the ratio (%) between 4G8⁺ Aβ immunoreactivity and α-actin⁺ pial vessel area obtained with ImageJ in the same sections in which smooth muscle fragmentation was quantified. However, the A β specie (1–40, 1–42) was not determined. Smooth muscle cell fragmentation was quantified by counting α-actin fragments in pial-penetrating arteriole using ImageJ, expressed as the fragmentation index: 100 − [(1/number of α-actin fragments) × 100].

Tangential brain sections were first incubated with α-actin (1:300, rabbit polyclonal; Abcam) or Glut-1 (rabbit polyclonal, 1:200, Calbiochem) antibody for 48 h, and, after washing, followed by Alexa 647-conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch). After mounting and drying on slides, brain sections were rehydrated with PBS and refixed with 4% PFA for 10 min. After washing, sections were labeled with 0.5% (wt/vol) thioflavin-S in 50% (vol/vol) ethanol for 10 min to identify CAA. Confocal images were obtained with an Alexa 488 filter for thioflavin-S and an Alexa 647 filter for α-actin or Glut-1. Images of α-actin or Glut-1 with thioflavin-S were acquired, and the number of α-actin⁺ or Glut-1⁺ was quantified. The CAA burden was expressed by the number of neocortical parenchymal vessels positive for both thioflavin-S and α-actin or Glut1 [20 (link), 30 (link)].

To quantify Aβ-associated fragmentation of smooth muscle cells in pial vessels [20 (link), 30 (link)], brain sections were incubated with anti-Aβ (4G8, 1:1,000, mouse; Covance) and the smooth muscle marker anti-α-actin (1:300, rabbit, Abcam) for 48 hr. After washing, sections were labeled with Alexa 488-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch) and Alexa 647-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch). Pial arterioles (n = 30–50/group) positive for Aβ and α-actin, ranging in diameter from 20 to 100 µm, were randomly imaged by confocal microscope (63x). The fragmentation of smooth muscles was quantified by counting the number of α-actin fragments of each arteriole using ImageJ, expressed as the fragmentation index: 100 − [(1/number of α-actin fragments) x 100].