Ab16794

Immunofluorescent (IF) double labeling was used to co-localize PPARγ in neurons, oligodendrocytes and astrocytes in the hippocampus as previously described (Fung et al. 2012 (link)). 49,6-diamidino-2-phenylindole (DAPI) was used for nuclei counter staining. The following primary antibodies were used: anti-PPARγ (#2435, Cell Signaling) labeled in red, anti-neuron marker NeuN antibody (MAB377; Millipore, Billerica, MA); anti- oligodendrocyte marker APC antibody (ab16794, abcam) and anti-astrocyte marker GFAP antibody (IF30L-100ug; Millipore, Billerica, MA). Negative control sections underwent similar staining procedures with the omission of primary antibody.
Sections were imaged with the Nikon’s A1 Nikon’s A1 multiphoton laser scanning confocal microscope (Nikon Instruments Inc./Americas) and taken at ×20 and ×60 magnification to encompass all subregions of the hippocampus (CA1, CA3, and DG).

Brain sections were stained with anti-CD68 (MCA1957GA, BioRad Laboratories, Hercules, CA, US), anti-CC1 (AB16794), anti-GFAP (AB4674), anti-neutrophil antibody 7/4-FITC (AB53453), and anti-vimentin (AB92547) (Abcam, Cambridge, MA, US), anti-Ki67-APC (50569882, Invitrogen, Carlsbad, CA, US), and anti-Iba1 (01919741, Wako Chemicals, Japan) antibodies overnight at room temperature. Meninges were stained with anti-neutrophil antibody 7/4-FITC (AB53453, Abcam, Cambridge, MA, US).
In the brain, left and right CA1 regions of the hippocampus were imaged. ‘Coloc 2’ was used to co-localize GFAP and Vimentin. For meninges, whole mounts of the meninges were stained for neutrophils (NT 7/4; AB53457, Abcam, Cambridge, MA, US). All images were obtained on the Olympus FV1200 confocal microscope with Fluoview software (Tokyo, Japan) and processed with ImageJ/Fiji (NIH, Bethesda, MD, US).
To quantify microglial activation, Sholl analysis was performed using brain sections labelled with anti-Iba1 to quantify the number of processes observed on selected cells using the protocol previously described ¹⁹ . To determine the inflammatory state of microglia, brain sections were co-labeled with Iba1 and CD68.