Gtx100042

After 3 months of WGE treatment, mice were sacrificed at the age of 11.5 months. The substantia nigra and striatum were dissected out. The substantia nigra was subjected to immunostaining, as described previously (Lin et al., 2020 (link)). Anti-tyrosine hydroxylase (TH) (Millipore, AB152, 1:200) and anti-ionized calcium-binding adapter molecule 1 (Iba-1) (GeneTex, GTX100042, 1:200) were used as primary antibodies for 24 hr at 4°C. Secondary antibodies were DyLight 488 goat anti-rabbit 1:300 and Alexa Fluor 546 goat anti-rabbit at 1:200 (25°C for 1 hr). Mounting medium with DAPI (GeneTex, GTX30920) was used as a counterstain.

Brain tissues were processed for immunofluorescent detection. The primary antibodies used were anti-ionized calcium-binding adaptor molecule-1 (Iba-1; 1:250; GTX100042, GeneTex, Alton Pkwy Irvine, CA, USA) and anti-MAP2 (1:200; GTX11267, GeneTex, Alton Pkwy Irvine, CA, USA). The brain sections were incubated with primary antibodies overnight, then washed and incubated with either Alexa Fluro 488- (1:500; GTX213111, GeneTex, Alton Pkwy Irvine, CA, USA) or Alexa Fluro 594- (1:500; GTX213110, GeneTex, Alton Pkwy Irvine, CA, USA) tagged secondary antibodies at room temperature. The number of cells positive for Iba-1 and MAP2 was counted in three non-overlapping fields in the cortex and hippocampus at ×200 magnification. Next, the sections were incubated in a 1:200 dilution of a TRITC-conjugated goat anti-rabbit secondary antibody and FITC-conjugated goat anti-mouse secondary antibody for 1 h at room temperature and then washed three times with PBS for 10 min. Then, the sections were stained with DAPI solution for 10 min at room temperature. Finally, fluorescence images were obtained by fluorescence microscopy (Leica DM 6000B; Mannheim, Germany), and the number of cells was counted with the ImageJ software.