Donkey anti goat igg

After deparaffinization and heat-mediated antigen retrieval (EDTA-based pH 9.0 solution), as previously described, the tissue sections were blocked with goat serum for 30 min at 37°C and then incubated at 4°C overnight with primary antibody (mouse anti-myelin basic protein (MBP) monoclonal antibody, 1 : 400, Abcam, USA; rabbit anti-synaptophysin monoclonal antibody, 1 : 100, Abcam, USA; goat anti-Olig2 polyclonal antibody, 1 : 50, R&D, USA). Sections were washed three times with 0.1 M phosphate buffered saline (PBS) for 5 min before incubation with secondary antibodies (donkey anti-rabbit IgG, 1 : 500, Abcam, USA; donkey anti-mouse IgG, 1 : 500, Abcam, USA; donkey anti-goat IgG, 1 : 200, Abcam, USA) for 4 h at room temperature and 4′,6-diamidino-2-phenylindole (DAPI; 1 : 500, Sigma-Aldrich) sequentially for nuclear staining. Immunofluorescent images were visualized and photographed using a confocal laser-scanning microscope (C1; Nikon, Tokyo, Japan).

Immunofluorescence staining on brain and cell slices was performed as previously described (8 (link), 12 (link)). Slices were treated with 0.3% Triton X-100 for 30 min and 10% donkey serum for 2 h, followed by incubation with primary antibodies at 4°C overnight. The primary antibodies were diluted with PBS as follows: rabbit anti-NeuN (1:300, Abcam), goat anti-GFAP (1:1000, Abcam), rabbit anti-p-p65 (S536) (1:100, CST), and rabbit anti-p65 (1:400, CST). The secondary antibodies (1:200, Abcam) were as follows: donkey anti-rabbit IgG (Alexa Fluor 594), donkey anti-goat IgG (Alexa Fluor 594), donkey anti-mouse IgG (Alexa Fluor 594), and donkey anti-rabbit IgG (Alexa Fluor 488). DAPI was obtained from Sigma (Saint Louis, MO, USA). Negative controls without primary antibody were performed for all samples.
TUNEL assay was used according to the manufacturer's protocol (Roche, Mannheim, Germany). Double-staining of TUNEL/NeuN was performed as previously described (22 (link)). In brief, the sections were initially stained with NeuN (1:1000, Abcam) and subsequently stained with TUNEL.
The sections were imaged using confocal microscopy. Five representative visual fields were randomly chosen to be analyzed, from the penumbra (Figures 7C,D) in each slice or from each dish. The fluorescent images were analyzed using Image-Pro Plus 6.0 or ImageJ by an observer who was blinded to grouping.