Sp 9002

Sections were stained for myelin with the use of luxol fast blue stain (LFB; Abcam) and for axons using immunohistochemical staining for phosphorylated neurofilaments (SMI-31; Abcam; Budde et al., 2007 (link)). Immunohistochemical staining for SMI-31 was performed as described previously (Budde et al., 2007 (link)). After accepting antigen retrieved with the use of citrate antigen retrieval solution (P0081; Beyotime), the tissue sections were treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. The sections were then incubated in 1:20 goat serum for 30 min, rinsed, and incubated overnight at 4°C with a 1:200 dilution of the primary antibody SMI-31. After three washes in PBS, the sections were incubated for 90 min with biotinylated goat anti-mice immunoglobulin G antibody (SP-9002; Zhongshan Golden Bridge Biotechnology). After another three PBS washes, the brain sections were incubated with avidin biotinylated horseradish peroxidase (SP-9002; Zhongshan Golden Bridge Biotechnology) for 90 min. The brain sections were rewashed three times in PBS and then incubated with diaminobenzidine and hydrogen peroxide (ZLI-9018; Zhongshan Golden Bridge Biotechnology). The nucleus was stained with hematoxylin. The sections were then rinsed in water for 10 min, dehydrated, and covered with a coverslip for microphotography.

Immunohistochemistry was performed as previously described [43 (link), 44 (link)]. The paraffin sections (5 μm) were deparaffinized in xylene, rehydrated with a graded series of ethanol, and washed in water. Antigen retrieval was performed by microwaving the sections in 10 mM sodium citrate buffer (pH 6.0) followed by cooling the sections to room temperature. The endogenous horse radish peroxidase activity was inhibited with 3% H₂O₂ for 15 min. After nonspecific binding was blocked with 10% horse serum at 37 °C for 1 h, the sections were incubated with primary antibody at 4 °C overnight. The primary antibodies used in this study include mouse anti-HSP27 antibody (1:800; ab79868, Abcam, Cambridge, UK), mouse anti-HSP70 antibody (1:800; ab5439, Abcam) and mouse anti-HSP90 antibody (1:400; ab13492, Abcam). The sections were then incubated with a biotin-labeled goat anti-mouse IgG antibody (1:1000; ab6789, Abcam) and a streptavidin-conjugated HRP complex (SP9002, Zhongshan Golden Bridge, Beijing, P. R. China). Finally, the signals were visualized with the DAB Horseradish Peroxidase Color Development Kit (ZLI9018, Zhongshan Golden Bridge) and counterstained with hematoxylin.

The paraffin sections (5 μm-thick) were deparaffinized in xylene, rehydrated with graded ethanols, and washed with water. Antigen retrieval was performed by microwaving the sections in 10 mM sodium citrate buffer (pH 6.0) and cooling them to room temperature. The endogenous horseradish peroxidase activity was inhibited with 3% H₂O₂ for 15 min. After the nonspecific binding was blocked with 10% horse serum at 37 °C for 1 h, the sections were incubated with primary antibody at 4 °C overnight. The primary antibodies used in this study include the mouse anti-Bcl-2 antibody (1:20; sc-7382, Santa Cruz Biotechnology (Shanghai) Co., LTD. China), and the mouse anti-Bax antibody (1:500; ab77566, Ai Bo Kang (Shanghai) Trading Co., LTD, China), and the mouse anti-LC3B antibody (1:200; ab229327, Ai Bo Kang (Shanghai) Trading Co., LTD, China), and the mouse anti-PINK (ab137361, Ai Bo Kang (Shanghai) Trading Co., LTD, China), and the mouse anti-Parkin (bs-23687R, Beijing Boaosen Biotechnology Co. LTD, China). The sections were then incubated with a biotin-labeled goat anti-mouse IgG antibody (1:1000; ab6789, Abcam) and a streptavidin-conjugated HRP complex (SP9002, Zhongshan Golden Bridge, Beijing, P. R. China). Finally, the signals were visualized with the DAB Horseradish Peroxidase Color Development Kit (ZLI9018, Zhongshan Golden Bridge) and counterstained with hematoxylin.