Alexa fluor 488 conjugated goat anti rabbit igg

The rats were killed by intraperitoneal injection (10% chloral hydrated, 1 ml/100 g) and 4% paraformaldehyde (PFA) was used to fix via cardiac perfusion. Coronal brain slices (15 μm thick) were prepared and used for staining. Slices were washed in PBS for 5 min, three times, then blotted in 5% goat serum for 1 h at room temperature. Slices were incubated with the primary antibodies overnight at 4°C. Primary antibodies used: mouse anti-Iba1 (1:200 Sigma American), mouse anti-GFAP (1:200 Sigma American), rabbit anti-iNOS, anti-Arg1, anti-IL-1β, anti-TNF-α, anti-IL-4, anti-IL-6, and anti-IL-10 (1:200; Bioss, Beijing, China). After having been washed in PBS three times, these slices were incubated with secondary antibodies at 37°C for 2 h. Secondary antibodies: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Bioss, Beijing, China), Cy3-conjugated goat ant-mouse IgG (1:200; Bioss, Beijing, China). Then these slices were washed three times again and stained with DAPI (Biyuntian, China) for 5 min. Results were imaged using a 7266-fluorescence microscope (Leica, Japan).

Sections were deparaffinized in xylene and rehydrated through graded alcohol series. After that, sections were washed three times in phosphate-buffered saline (PBS) for 5 min each, then incubated with 5% goat serum for 1 h at 37 °C. Sections were incubated with the rabbit anti-PPAR-γ, anti-iNOS (1:100, Proteintech, Wuhan, China), rabbit anti-Arg1 (1:200, Bioss, Beijing, China), rabbit anti-Aβ_1–42, anti-P-Tau (1:200, Abcam, Waltham, MA, USA), mouse anti-Iba1 and anti-F4/80 (1:200, Bioss, Beijing, China) overnight at 4 °C. The sections were incubated with Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (1:200, Proteintech, Wuhan, China) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Bioss, Beijing, China) at 37 °C for 2 h in a dark room after being washed 3 times with PBS. In the final step, the sections were rinsed 3 more times before being stained for 5 min with DAPI (Biyuntian, Shanghai, China). The sections were imaged using Pannoramic MIDI (3DHISTECH, Budapest, Hungary).

UECM samples on plastic slides were fixed with 4% paraformaldehyde in PBS for 30 min. After incubation in 10% normal goat serum for 30 min at room temperature, the samples were treated with primary antibodies against type I collagen (Abcam, UK), fibronectin (Abcam), and laminin 5 (Abcam) at 4 °C overnight, followed by incubation in Alexa Fluor 488-conjugated goat anti-rabbit IgG (Bioss, China) and Alexa Fluor 647-conjugated goat anti-mouse IgG (Bioss) for 30 min. Images were recorded by confocal laser scanning microscopy (Leica TCS SP8 X, Germany).