Anti gfap primary antibody

For Western blot analysis, five male animals from the control and experimental groups (P60) were killed by cervical dislocation and decapitation without anesthesia. After decapitation, the brains were quickly removed and transferred to liquid nitrogen. The dorsolateral frontal cortex (4.2 mm up to −1.32 mm from bregma; [38 ]) was homogenized in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail) on ice for 30 min, centrifuged at 18.000 g for 15 min at 4°C, and the supernatant was collected. An equal amount of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel and transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK). Rabbit polyclonal anti-GFAP primary antibody (Dako, Denmark) was used. All membranes were stripped and reprobed with anti-actin antibody (Sigma-Aldrich) to ensure equal loading. Western blots were scanned and densitometric analysis was performed using ImageQuant 5.2 (GE Healthcare, Buckinghamshire, UK).

Paraformaldehyde-fixed brains were sectioned coronally (at 20 μm thickness), using a sliding microtome (CM1900; Lieca, Nussloch, Germany). For immunohistochemistry (IHC), slices were blocked with phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) and 0.5% triton X-100 for 1 hour, incubated overnight with anti-GFAP primary antibody (Z0334; Dako Cytomation, Glostrup, Denmark) and anti-synaptophysin antibody (04–1019, Millipore), at 4 °C, and then in DyLight 488-AffiniPure anti-rabbit IgG secondary antibody (111–485–003; Jackson ImmunoResearch, Westgrove, PA, USA) for 1 hour. For Thioflavine-S staining, slices were stained with 0.015% Thioflavine-S (T1892; Sigma, MO, USA) for 15 minutes at room temperature. After mounting, slides were imaged using a Zeiss fluorescence microscope (Axio Observer A1; Zeiss, Oberkochen, Germany).

Formaldehyde fixed tissue samples were processed using the ExcelsiorAS system (ThermoScientific) according to the manufacturer’s recommendations. For GFAP immunohistochemistry a ready to use anti-GFAP primary antibody (Dako, Hamburg, Germany) was used and the samples were processed on an automated system, EnVision™ FLEX (Dako), following manufacturer’s instructions. Hematoxylin and Eosin staining was performed following established standard protocols [25 ].