Goat anti guinea pig igg alexa fluor 594

Animals were euthanized by cervical dislocation; brains were removed, and the right hemisphere was immediately fixed in 4% paraformaldehyde (PFA) at 4 °C overnight and embedded in paraffin for histological analyses. Five-micrometer paraffin sagittal sections were obtained by microtome and sections were deparaffinized and hydrated with deionized water. The detection of PGs by periodic acid Schiff (PAS) staining and immunohistochemical analysis were performed as detailed in [29 (link)]. For immunohistochemistry, primary and secondary antibodies used were guinea pig anti-GFAP (1:500, Synaptic Systems #173_004), rabbit anti-Iba1 (1:200, WAKO #019–19741), Alexa Fluor-conjugates [1:500, Life Technologies: goat anti-guinea pig IgG Alexa Fluor® 594 (#A11076)], and goat anti-rabbit IgG [Alexa Fluor® 488 (#A11008)]. Background controls of secondary antibodies were performed in parallel. Nuclear staining was performed with DAPI (Sigma-Aldrich). Coverslips were mounted with Fluoromount-G™ (Thermo Fisher Scientific).

Brain slices were prepared according to the procedures mentioned above. The selected brain slices were incubated in 0.1% Triton X‐100 for 0.5 hour and then blocked with PBS solution containing 5% goat serum (Gibco) for 30 minutes. The slices were then incubated at 4°C for 12 hours with primary antibodies—anti‐p‐Smad3 (1:200, ab52903, Abcam), anti‐NeuN (1:500, APN90P, Millipore). After being rinsed with PBS, the slices were incubated with corresponding secondary antibodies—donkey anti‐rabbit IgG (Alexa Fluor 488, Life Technologies), goat anti‐guinea pig IgG (Alexa Fluor 594, Life Technologies)—for 60 minutes at 25°C. Next, sections were stained using DAPI (Invitrogen) for 15 minutes at 25°C. In FITC‐labelled APNp (Sangon Biotech Co.) treated group, mice brain slices were directly incubated with DAPI for 15 minutes at 25°C. Finally, all sections were analysed with a fluorescence microscope (A1 Si, Nikon) by independent observers blinded to the experiment.