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URL: https://dx.doi.org/DOI:10.17504/protocols.io.mmpc45n

Authors: Ning Ding

Summary: Forty SD male rats were divided into five groups randomly (n = 8 per group): the normal control (N) group, the normal saline group (NS), the light osteoarthritis group (A), the mild osteoarthritis group (B) and the heavy osteoarthritis group (C). On day 0, the rats in the A, B and C group were injected with 0.3, 1, 3 mg MIA respectively, and rats in the NS group were injected with 50 μl of saline solution.The tissues at the acupoints Yanglingquan (GB34), Heding (EX-LE2) and Weizhong (BL40) were dissected. The dimensions of each tissue fragment were 1.5 × 1.5 × 1.5 mm. The tissues were fixed in 10% neutral formalin, dehydrated, embedded in paraffin and sectioned with a 4-μm slicer. Subsequently, the sections were dewaxed, dehydrated and stained in 0.5% toluidine blue for 30 min and then washed with tap-water.the knee joints of 6 rats in each group were fixed in 10% neutral formalin after samples of GB34, EX-LE2 and BL40 were dissected. Then, the knee joints were placed in 5% formic acid for demineralization, dehydrated, embedded in paraffin and sectioned with a 5-μm slicer. Subsequently, the sections were dewaxed, dehydrated, and stained in 1% fast green for 1.5 min and then differentiated by acetic acid. Next, the sections were stained by 0.5% safranine-O for 1.5 min, differentiated by ethanol, and washed with tap-water. The tissues at GB34, EX-LE2, and BL40 were dissected, fixed in 4% paraformaldehyde for 2.5 h, and dehydrated by 25% sucrose for 2 days. A frozen 10-μm section was sliced by a freezing microtome. For staining, the sections were washed in 0.1 M PB (pH=7.4) and blocked in 0.1 M PB (pH=7.4) containing 3% normal donkey serum and 0.5% Triton X-100 for 30 min. Next, the sections were treated with mouse monoclonal mast cell tryptase antibody and goat polyclonal serotonin antibody and incubated overnight at 4℃. After PB washes, the sections were exposed to Donkey Anti-Mouse IgG Alexa Fluor 488 and Donkey Anti-Goat IgG Alexa Fluor 594 for 2 hours. Subsequently, the sections were washed with PB and stained with DAPI for 6 minutes. After washing with PB, the sections were observed under a confocal laser scanning microscope. Double immunohistochemical staining using the same staining protocol was undertaken, to examine the co-expression of mast cell tryptase and HA.

Associated Publications: Ding N, Jiang J, Qin P, Wang Q, Hu J, Li Z (2018) Mast cells are important regulator of acupoint sensitization via the secretion of tryptase, 5-hydroxytryptamine, and histamine. PLoS ONE 13(3): e0194022. doi: 10.1371/journal.pone.0194022

Affiliations: Beijing University of Chinese Medicine

External URL: https://doi.org/10.1371/journal.pone.0194022

Version: 1

Publication Date: 2018

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