改良骨骼肌肌纤维分型ATP酶染色方法

Modified procedure for the histochemical demonstration of myosin ATPase

目的采用经作者改良的Guth-Samaha肌球蛋白ATP酶染色方法,观察其对大鼠骨骼肌肌纤维分型染色效果。方法取8μm厚度SD大鼠胸肌及肱二头肌冰冻切片,用含40 g/L多聚甲醛固定液固定5 min;Tris洗液漂洗后置预孵育液中室温下孵育15 min;Tris洗液中漂洗2次,置孵育液中于37℃恒温水浴摇床中振摇孵育60 min;10 g/L氯化钙洗3次后置20 g/L氯化钴液中3 min;蒸馏水冲洗,于10 g/L硫化铵液中孵育3 min;自来水冲洗3 min,常规梯度乙醇脱水,二甲苯透明,中性树胶封片,置显微镜下观察。结果SD大鼠胸肌和肱二头肌经改良Guth-Samaha肌球蛋白ATP酶染色后均可清晰地显示出明亮色白的Ⅰ型纤维和幽暗深褐的Ⅱ型纤维。结论改良Guth-Samaha肌球蛋白ATP酶染色方法具有试剂配制简单、操作简化和肌纤维分型效果满意等优点,值得在骨骼肌相关研究中借鉴应用。

Objective To study the effect of self-modified Guth ＆ Samaha＇s myosin-ATPase staining method on the classification of SD rat skeletal muscle fiber types. Methods 8μm-thick cryostat sections from rat chest muscles and biceps brachii muscles were transfected with cryostat. Myosin-ATPase staining was carried out according to the following procedure： ① Fixing sections for 5 min in fixative solution containing 40 g/L paraformaldehyde; ② Rinsing slides in Tris-rinse solution and then preincubating them in alkaline preincubation solution for 15 rain; ③ Rinsing slides in Tris-rinse solution twice and then incubating them for 60 min in incubation solution; ④ Washing slides for three times in 10 g/L CaCl2 and then placing them in 20 g/L CoCl2 for 3 min; ⑤ Washing sides in distal water and then placing them in 10 g/L （NH4）2S for 3 min and; ⑥ Washing slides in running tap water for 3 min, dehydrating in graded ethanol, clearing in xylene and mounting in neutral balsam. Results After being stained by modified myosin-ATPase staining method, both chest muscle and biceps brachii muscle samples from SD rats could be clearly identified as type Ⅰ fibers and type II fibers as the fibers of type Ⅰ were stained white while the fibers of type Ⅱ were stained brown. Conelusion Modified myosin-ATPase staining method is a simple and effective way for muscle fiber type classification and can be applied in skeletal muscle related study.