Ab3558

The immunohistochemical experiments were conducted as previously described [25 (link)]. The antibodies used in this study included an anti-Iba1 antibody (1:50; ab178846; ab283319; Abcam, MA, USA), an anti-CD86 antibody (1:200; ab220188; Abcam, MA, USA), anti-IL-1β antibody (1:200; ab254360; Abcam, MA, USA), anti-TNF-α antibody (1:150; ab1793; Abcam, MA, USA), anti-IL-6 antibody (1:200; ab290735; Abcam, MA, USA), anti-ERα antibody (1:200; ab32063; Abcam, MA, USA), anti-GPER antibody (1:200; PA5-77396; Invitrogen, Karlsruhe, Germany), anti-ERβ antibody (1:150; PA1-311; Thermo Fisher Scientific, Shanghai, China), anti-NF-κB p65 antibody (1:200; ab32536; Abcam, MA, USA), anti-COX1 antibody (1:500; ab109025; Abcam, MA, USA), anti-CB1 antibody (1:200; ab3558; Abcam, MA, USA), anti-IL-1 (1:200; ab254360; Abcam, MA, USA), Alexa Fluor 488 secondary antibody (1:1,000; Invitrogen, Karlsruhe, Germany), Alexa Fluor 594 secondary antibody (1:1,000; Invitrogen), and IgG H&L (HRP) antibody (1:500; ab97051; Abcam, MA, USA). The nuclei were stained with DAPI (Sigma-Aldrich, Beijing, China) and images were captured on the LSM700 laser scanning confocal microscope (Zeiss, Tokyo, Japan) with the mean fluorescence intensities (MFI) determined using the FlowJo software [26 (link)].

The left cerebral hemispheres were fixed in 4% PFA, dehydrated through a series of sucrose solutions, and sectioned at 25 µm thickness. The brain sections were incubated overnight at 4 °C with the primary antibodies (1:100 dilution for all) specific to the protein of interest including Aβ (developed in-house), total tau (ab76128, Abcam, Cambridge, MA, USA), phospho-tau (ab92676, Abcam, Cambridge, MA, USA), GSK3β (12456, Cell Signaling Technology, Danvers, MA, USA), Iba-1 (ab178846, Abcam, Cambridge, MA, USA), neuronal nuclei (NeuN) (ab177487, Abcam, Cambridge, MA, USA), and CB1 (ab3558, Abcam, Cambridge, MA, USA). After incubation with the primary antibody, the brain sections were subjected to a 1-h incubation with the biotinylated secondary antibody according to the manufacturer’s protocol (Vector Laboratories). Light microscopy staining was achieved with the standard biotin-streptavidin/HRP procedure and DAB chromogen as described elsewhere [80 (link)]. The sections were then counterstained with hematoxylin and mounted under coverslips. For each protein of interest, three sections were selected from the same hippocampus layer of the brain and used for analysis. All three measurements were averaged for each mouse to yield the value for further statistical analysis. The abnormal overstained or cracks were excluded from the analysis field.