Glial fibrillary acidic protein (gfap)

The brain tissues were frozen in Surgipath FSC 22 clear frozen section compound (Leica Microsystems) using dry ice and isopentane. The harvested brain tissues were cryosectioned into 16-μm-thick sections along the coronal plane, and immunohistochemical staining was performed as previously described. Eight weeks after EE, to confirm the endogenous expression of MAP2 (1:400, Abcam), GFAP (1:400, Neuromics), MCT2 (1:400, Santa Cruz), and MCT4 (1:400, Santa Cruz), the brain sections of the cerebral cortex and hippocampus were immunostained. The sections were incubated with Alexa Fluor^® 488 goat anti-rabbit (1:400, Invitrogen) and Alexa Fluor^® 594 goat anti-mouse (1:400, Invitrogen) secondary antibodies, and then covered with Vectashield^® mounting medium with 4C, 6-diamidino-2-phenylindole (Vector, Burlingame, CA, USA). The stained sections were analyzed using an LSM 700 confocal microscope (Zeiss, Gottingen, Germany). The z-stack confocal analysis was used to measure the colocalization of MAP2 with MCT2 and GFAP with MCT4 and to create Maximum Intensity Projection (MIP) and Ortho (2.5D) images for further colocalization clarification.

Sections were rehydrated in 1 × PBS (3 × 5 min) and then incubated with 10% normal goat serum for 30 min with 0.1% triton-X. To measure astrocyte reactivity, sections were incubated overnight at room temperature in polyclonal anti-glial fibrillary acidic protein (GFAP) (Neuromics, MN, USA; #CH22102; 1:500). Images were taken with a Leica DFC340 FX (Leica, Germany) at three different locations for each brain region and the percent area above fluorescence threshold was averaged for each animal. The same settings were used for all sections. Microglial migration and reactivity as well as infiltrating macrophages were assessed using the polyclonal anti-Iba-1 (Wako, VA, USA; #019-19741; 1:1000). After rinsing with 1 × PBS, sections were then incubated with Alexa 546 conjugated secondary antibodies (Invitrogen, USA; #A-11035; 1:250) for 1 h at room temperature. Sections were mounted with ProLong Gold antifade (Invitrogen, USA; #P36934).

Cells or cryosectioned tissues (10 μm) were post-fixed with 4% paraformaldehyde at room temperature for 10 min. After permeabilization with PBS containing 0.5% Triton and 2% goat serum (blocking solution), the primary antibodies were incubated overnight at 4 °C. Cells or tissue sections were incubated with GAP43 (α-rabbit; Cat. ab128005 Abcam, Hong Kong, China), β-tubulin III (α-mouse; Cat. MO15052 Neuromics, Edina, MN, USA), GFAP (α-rabbit; Cat. Z0334 DAKO, Santa Clara, CA, USA), RIP (α-mouse; Cat. MAB1580 Chemicon, Pittsburgh, PA, USA) and OLIG1 (α-rabbit; Cat. AB5320 Chemicon). Primary antibodies were diluted 1:200 in blocking solution. After being rinsed three times with PBS, the cells or the tissue sections were incubated with Oregon Green-Alexa488, Alexa555 or Alexa647 dye-conjugated secondary antibodies for 1 h at room temperature. All cells and tissue sections were counterstained by incubation with DAPI for 5 min at room temperature followed by washing steps. Signals were visualized by both fluorescent microscopy (fluorescence microscope Leica DM6000B, Wetzlar, Germany) and confocal microscopy (confocal microscope Leica TCS-SP2-AOBS, Wetzlar, Germany). The quantification of immunostainings was performed using ImageJ software (Image J2, Madison, WI, USA).