Goat anti mouse or goat anti rabbit igg h l

The sections were dried on a slide warmer and incubated with blocking buffer (5% BSA) for 1 h at 25 °C, washed with blocking buffer. Diluted primary antibodies against neuronal nuclear (NeuN), glial fibrillary acidic protein (GFAP), the cluster of differentiation 68 positive (CD68+) (Cat. no. 94403, 12389, and 97778, respectively (Cell Signaling Technology, Danvers, MA, USA), and tumor necrosis factor-alpha (TNF-α) (ab1793, Abcam, Cambridge, UK) were incubated with the sections overnight at 4 °C. The primary antibodies were washed three times with PBS for 5 min, and the diluted secondary antibodies (goat anti-mouse or goat anti-rabbit IgG H&L, Abcam, Cambridge, UK) were dropped on the sections at 25 °C for 2 h. The secondary antibodies were washed three times with PBS for 5 min each. After dropping the mounting medium with DAPI (ab104139, Abcam, Cambridge, UK), the sections were covered with cover slides, and the edges were sealed with nail polish. After observation under a fluorescence microscope (Ni-U, Nikon, Tokyo, Japan), the samples were stored in a refrigerator at 4 °C for long-term storage. The degree of color intensity in response to fluorescence after staining with an antibody was regarded as the expression level of the protein, and the fluorescent intensity was analyzed using ImageJ (NIH, Bethesda, MD, USA).

The sections were dried in a slide warmer (Convision Co., Seoul, Korea), incubated with blocking buffer (5% BSA) for 1 h at 25 °C, and then washed with blocking buffer. Diluted primary antibodies against the neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) (catalogue numbers 94403 and 12389, respectively; Cell Signaling Technology, Danvers, MA, USA) and the tumour necrosis factor-alpha (TNF-α) (ab1793, Abcam, Cambridge, UK) were incubated with the sections overnight at 4 °C. The primary antibody was then washed off 3 times for 5 min with PBS, and the diluted secondary antibody (goat anti-mouse or goat anti-rabbit IgG H&L; Abcam, Cambridge, UK) was dropped onto the sections and incubated at 25 °C for 2 h; the sections were then washed 3 times with PBS for 5 min each. After mounting using DAPI (ab104139, Abcam, Cambridge, UK), the sections were mounted with cover slips, with the edges sealed with nail polish and observed under a fluorescence microscope (Ni-U, Nikon, Tokyo, Japan). The samples were stored in a 4 °C refrigerator for long-term use. After staining with antibodies, the colour intensity reacting to fluorescence was regarded as the protein expression level.