Sc 166458

Tissue sections were sliced into adhesive slides (4–5 µm thick). After rehydration and heat-induced epitope retrieval, the slides were washed, blocked in BSA and hydrogen peroxide, and then incubated overnight at 4 °C in a humid atmosphere with primary mouse anti-rat antibodies for myelin basic protein (MBP; sc-271524, Santa Cruz Biotechnology, Inc., dilution 1:200) or glial fibrillary acidic protein (GFAP; sc-166458, Santa Cruz Biotechnology, Inc., dilution of 1:200). The excess primary antibody was then washed off, and a secondary antibody labeled with HRP (ab97023- Goat anti-mouse HRP labeled antibody, 1:200) was applied for two hours at room temperature, followed by washing. Lastly, the reaction was observed under a light microscope using a DAB-substrate mix (Pierce^™ DAB Substrate Kit (34,002) from Thermo Scientific, Inc.). Negative control slides were created by omitting the primary antibody. MBP and GFAP expression was quantified as an area percentage of positive expression in 10 microscopic fields to obtain the mean value in each group using CellSens Dimensions software (Olympus, Japan).

Immunohistochemical staining of GFAP, IbA-1, and Aβ was conducted according to the manufacturer’s protocols and directions. First, antigen-retrieved brain sections were blocked with 3% hydrogen peroxide in methanol for 15 min and then incubated overnight at 4 °C with the primary antibodies (antiglial fibrillary acidic protein as a monoclonal antibody (sc-166458, Santa Cruz Biotechnology, Inc., Dallas, TX, USA, dilution of 1:200), anti-Iba1 antibody (ab108539-Abcam, 1:100), and anti-Aβ antibody (ab201060-Abcam, 1:500)). Next, the sections were washed with PBS three times and incubated with a secondary antibody HRP Envision kit (DAKO) for 20 min. Then, they were washed with PBS three times, incubated with diaminobenzidine for 10 min, counter-stained with Mayer’s hematoxylin, then dehydrated and cleared in xylene. Finally, samples were cover-slipped for microscopic examination.

Both tissue preparation and immunohistochemical examination were carried out. Briefly, after the rehydration process, epitope retrieval was performed with heat. The tissue sections were blocked with bovine serum albumin and hydrogen peroxide. A primary anti-mouse antibody against glial fibrillary acidic protein (GFAP) (sc-166458, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, dilution of 1:200) was incubated overnight in a humidified chamber at 4 °C. After a washing step to remove excess primary antibody, a secondary HRP-labeled antibody was applied (ab97023- Goat anti-mouse HRP-labeled antibody, Abcam, Cambridge, UK, 1:200) and incubated at room temperature 25 °C for 2 h. After washing, 3,3′-Diaminobenzidine (DAB) Substrate (Pierce™ DAB Substrate Kit (34002), Thermo Scientific, Inc., Rockford, IL, USA) was used to develop the reaction, which was examined by light microscopy. Negative control slides were prepared by removing the primary antibody incubation step. Images were captured with cell Sens Dimension software, Olympus, Tokyo, Japan, to measure the area occupied by GFAP-positive cells.