Forty\u00a0SD male rats were divided into five groups randomly (n = 8\u00a0per group): the normal control (N) group, the normal saline\u00a0group (NS), the light osteoarthritis group (A), the mild osteoarthritis group (B) and the heavy osteoarthritis group (C).\u00a0On 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 \u03bcl of saline solution.The\u00a0tissues at the acupoints Yanglingquan (GB34), Heding (EX-LE2) and Weizhong (BL40) were dissected.\u00a0The dimensions\u00a0of each tissue fragment\u00a0were\u00a01.5 \u00d7 1.5 \u00d7 1.5 mm. The tissues were fixed in 10% neutral formalin, dehydrated, embedded in paraffin and sectioned with a 4-\u03bcm slicer. Subsequently, the sections were dewaxed, dehydrated and stained in 0.5% toluidine blue for 30 min and then washed with\u00a0tap-water.the knee joints of 6 rats in each group were fixed in 10% neutral formalin after samples\u00a0of GB34, EX-LE2 and BL40 were dissected. Then, the knee\u00a0joints\u00a0were placed\u00a0in 5% formic\u00a0acid for\u00a0demineralization, dehydrated, embedded in paraffin and sectioned with a 5-\u03bcm slicer. Subsequently, the sections were dewaxed, dehydrated, and stained in 1% fast green for 1.5 min\u00a0and\u00a0then differentiated by acetic acid. Next, the sections were stained by 0.5% safranine-O for 1.5 min, differentiated by ethanol, and washed with\u00a0tap-water.\u00a0The tissues at GB34, EX-LE2,\u00a0and BL40 were dissected, fixed in 4% paraformaldehyde for 2.5 h,\u00a0and dehydrated by 25% sucrose for 2 days. A frozen 10-\u03bcm section was sliced\u00a0by\u00a0a\u00a0freezing microtome. For\u00a0staining,\u00a0the 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\u00a0mouse monoclonal mast cell tryptase antibody and goat polyclonal serotonin antibody and incubated overnight at 4\u2103. 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\u00a0with PB and stained with DAPI for 6 minutes. After\u00a0washing with PB, the sections\u00a0were observed\u00a0under a confocal laser scanning microscope. Double immunohistochemical staining\u00a0using the same staining protocol was undertaken,\u00a0to examine the co-expression of mast cell tryptase and HA.