Ab15690

Anti-PRDX6 immunohistochemistry was performed as previously described^{2 (link)} on sections of rat cerebral cortex (paraformaldehyde-fixed) using rabbit anti-PRDX6 antiserum (GWB-007PE6; GenWay Biotech; 1:250 dilution), or mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (MAB360; Millipore; 1:1000) for co-localization to astrocytes. Alexa Fluor^® 488 donkey anti-rabbit (green) or Alexa Fluor 488 donkey anti-mouse (red) detection antibodies (Invitrogen; 1:100 dilutions) were used to visualize PRDX6 and GFAP, respectively. Additional analyses of cellular co-localization were carried out using antibodies directed against Iba-1 (ab15690; Abcam, Cambridge, Massachusetts, 1:1000) and ED-1 (MCA341R; AbD Serotec, Raleigh, North Carolina; 1:150) for quiescent and activated microglia, respectively; and NeuN (MAB377; Millipore; 1:1000) for neurons. These data (not shown) confirmed that PRDX6 is primarily associated with astrocytes in rodent brain.

After the slides were pretreated as described in the immunohistochemical analysis section, the sections were simultaneously incubated with two primary or secondary antibodies. The primary antibodies used in the staining were as follows: mouse anti‐HCMV pp65 monoclonal antibody (ab49214, 1:200, Abcam, Cambridge, UK), rabbit anti‐IFITM3 polyclonal antibody (HPA004337, 1:200, Sigma‐Aldrich, St. Louis, MO, USA), mouse anti‐NeuN polyclonal antibody (ab104224, 1:200, Abcam, Cambridge, UK), mouse anti‐GFAP polyclonal antibody (3670s, 1:200, CST, Danvers, USA), mouse anti‐Iba‐1 polyclonal antibody (ab15690, 1:200, Abcam, Cambridge, UK), and mouse anti‐IE1 (ab65104, 1:40, Abcam, Cambridge, UK). The secondary antibodies were Alexa Fluor 488‐labeled donkey anti‐mouse immunoglobulin G (IgG) and Alexa Fluor 594‐labeled donkey anti‐rabbit IgG, 1:1000 for both (Life Technologies, USA). The nucleus was stained with DAPI, and the images were observed and recorded under a microscope (Olympus BX61, Olympus, Japan).

The cellular location of Ubqln was also determined in the brains of HI pups. Frozen sections were heated and washed with PBS then incubated with blocking solution with 10% normal goat serum for 1 h at room temperature. The sections were then incubated with primary antibody against RNA binding protein fox-1 homolog 3 (NeuN) for neurons (dilution, 1:1,000; cat. no. SAB4300883; Sigma-Aldrich; Merck KGaA), glial fibrillary acidic protein (GFAP) for astrocytes (dilution, 1:1,000; cat. no. ab10062, Abcam, Cambridge, USA), allograft inflammatory factor 1 (Iba-1) for microglial cells (dilution, 1:1,000; cat. no. ab15690, Abcam) and Ubqln (dilution, 1:500; cat. no. SAB1305680, Sigma-Aldrich, Merck KGaA) in PBS containing 0.3% Triton X-100 at 4°C overnight. The sections were then washed with PBS and incubated with the corresponding fluorescence-conjugated secondary antibodies (Alexa Fluor^®488 goat anti-mouse IgG (H+L), cat. no. A-11029; Alexa Fluor^®594 goat anti-rabbit IgG (H+L), cat. no. A-11012; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Nuclei were stained for DAPI (0.1 µg/ml), for 5 min at room temperature. Images were obtained using a fluorescence microscope (BX51; Olympus Corporation). Ischemic ipsilateral cortical and hippocampal regions were selected for imaging, and five visual fields were selected for each section (magnification, ×100 and ×200).

Mouse anti-TLR2 monoclonal antibody [T2.5] (ab59711), mouse anti-Iba1 monoclonal antibody (ab15690; used as marker for microglia) and mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (ab10062) were purchased from Abcam (Cambridge, UK); rabbit anti Sphk1 polyclonal antibody (sc-48825) and rabbit anti-TLR2 polyclonal antibody (sc-10739) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); fluorescence fluorescein isothiocyanate (FITC)-labeled goat anti-mouse secondary antibody (ZF-0312) and fluorescent rhodamine-labeled goat anti-rabbit secondary antibody (ZF-0316) were purchased from Beijing Zhongshan Golden Bridge Technology Co. (Beijing, China); the Ultrapure RNA extraction kit (CW0581), HiFi-MMLV cDNA First Strand synthesis kit (CW0744), UltraSYBR Mixture (with Rox) (CW0956), and DNase I (CW2090A) were purchased from Beijing Kangwei Century Biotechnology Co. (Beijing, China); immunoassay kits (IL-23, IL-17, IL-1, TNF-α, no. 870257) were purchased from LightArray Biotech Co. (Shanghai, China); and DMS (62575) was purchased from Cayman Chemical (Ann Arbor, MI, USA).