Ab26113

The procedure has been reported previously in [26 (link)]. Briefly, the mouse brains containing the nucleus raphe magnus (NRM) were harvested and immediately embedded in O.C. T. compound in liquid phlegm after the mice were sacrificed. The NRM sections were between 5.68 and 6.48 mm to the bregma and the NRM was located 1.72–2.68 mm interaurally, as described previously [26 (link)]. Serial 6-μm sections were performed on a cryostat for each block and stored at the temperature below −20 °C until use. The sections were incubated in goat anti mouse serum (BioSunTechnclogy) for 10 min and then incubated in mouse anti primary antibody against GAD65 (ab26113, Abcam; 1:1,000) at 4 °C overnight. GAD65 is expressed in the cytoplasm of presynaptic neurons. The sections were rinsed with PBS (pH 7.4) and incubated in secondary antibody mixed with DyLight649 (E032610, Earthox, San Francisco, USA) for one hour, and then rinsed with PBS (pH 7.4) again and mounted.
Images were obtained with a microscope (Olympus BX51) fitted with a digital camera (Olympus DP70). Five randomly selected images out of 6–7 sections of each mouse were taken for each immunostained parameter to count the numbers of GAD65-positive (red) cells in the NRM, located between 1.72 mm and 2.68 mm interaurally, and the mean was calculated.

The 18 μm brain slices were dried at 42 °C, hydrated by PBS, permeabilized and blocked with 5% BSA in PBST(PBS with 0.5% triton) incubated with primary antibody (goat anti-TNFα 1:200 R&D systems AF-410-NA; goat anti-TMEM119 1:200 abcam ab209064; rabbit anti-AMPAR1 1:100 abcam ab183797; rabbit anti AMPAR2 1:600 abcam ab206293, mouse anti-GAD65 1:200 abcam ab26113) at 4 °C overnight. For secondary antibody, delight donkey anti goat 488(earthox E032231 1:200), delight donkey anti rabbit 594(earthox E032421 1:200), delight goat anti mouse 488(earthox E032210 1:400), delight goat anti rabbit 594(earthox E032420 1:400)were used.

At 3 h after the KA injection for acute experiments, rats were transcardially perfused with saline, and their brains were then dissected and fixed with 4% paraformaldehyde for 24 h at 4 °C. Brain tissue was embedded in paraffin and sectioned into 6-µm-thick coronal planes at −3.0 mm AP. To conduct histological analysis of each group, Nissl staining (Nissl Stain Kit, IHC WORLD, UK) and immunohistochemistry with antibodies including c-Fos (ab209794, dilution 1:200, Abcam, UK), GAD65 (ab26113, dilution 1:100, Abcam), and Iba1 (ab178846, dilution 1:2000, Abcam) were performed on brain sections. Prior to c-Fos staining, pH 6.0 citrate retrieval (ZUC028-500, Zytomed, Germany) treatment was performed at 121 °C for 7 min. Anti-mouse (K4001, dilution 1:2000, Agilent DAKO, USA) was used as the secondary antibody for c-Fos and GAD65, and anti-rabbit (K4003, dilution 1:2000, Agilent DAKO) was used as the secondary antibody for Iba1. The stained slides were scanned using an Aperio ScanScope AT system (Leica Biosystems, USA), and then captured the CA1, CA3, DG, and cortex area using the Aperio ImageScope software (Leica Biosystems). Brain tissue with broken brain anatomical landmarks was excluded from the quantitative analysis. ImageJ (National Institutes of Health, USA) was used to measure the number of positive cells.