论文部分内容阅读
NADPH—d组化染色是定位NOS神经元的流行方法。FOS表达与NOS激活具有共同的上游事件，即Ca2+内流、钙调素依赖机制，FOS蛋白与NOS是否存在于同一种神经元中尚缺乏形态学方面的证据。F。S是一种核蛋白，定位在神经元核内，免疫组化染色后呈褐色或棕色，NADPH—d定位在胞浆中，组化染色呈现兰色，这为两种方法结合提供了良好的双染条件。结合途径有两种①先染NADPH－d再染FOS；②先染F。S再染NADPH－d疽种方法我们及其它作者已有报道。但在Soman诱发惊厥大鼠模型中，对杏仁核、下丘脑染色未能取得满意效果。因此，我们试用了另一结合途径。结果显示了满意的双染效果。现将这一方法介绍如下：1．ABC免疫组化：常规ABC免疫组化：我们采用低温、高抗体稀释度、长时间孵育方法，DAB呈色。2．切片入恢复液；配方是0IMPB配制，含0．05MCaCI。、0．of％双氧水，O25％TritonX－10OI。37C，30min。3．入NADPH—d染液：0ling／mlNBTling／mlNADPH-d。0．3％Tritonxl0oin0．IMPBpH7．337C40～6Omin，这一染色方法成功率高，双染效果好，色泽鲜艳。这一方法尤其适用于NADPH－d神经元大小中等，且比较集中的脑区进行染色。应用这一方法应该注意的是：①应尽量减少免疫组化染色的背底；②37℃孵育时；司不直超过Zh，③只
NADPH-d histochemical staining is a popular method of targeting NOS neurons. FOS expression and NOS activation have common upstream events, that Ca2 influx, calmodulin-dependent mechanism, FOS protein and NOS exist in the same kind of neurons is still lack of morphological evidence. F. S is a nuclear protein located in the nucleus of neurons, brown or brown after immunohistochemical staining, NADPH-d located in the cytoplasm, histochemical staining showed blue, which provides a good combination of the two methods Double staining conditions. There are two ways of combining ① first dye NADPH-d and then FOS; S Re-dyed NADPH-d gangrene method We and other authors have reported. However, in Soman-induced seizures rats model, the amygdala, hypothalamic staining failed to achieve satisfactory results. So we tried another way of combining. The results show a satisfactory double-dye effect. Now this method is introduced as follows: 1. ABC immunohistochemistry: conventional ABC immunohistochemistry: We used low temperature, high antibody dilution, long incubation method, DAB color. 2. Slice into the recovery fluid; formula is 0IMPB preparation, containing 0.05MCaCI. , 0. of% hydrogen peroxide, O25% TritonX-10OI. 37C, 30min. 3. Into the NADPH-d dye: 0ling / mlNBTling / mlNADPH-d. 0.3% Tritonxl0oin0. IMPBpH7.337C40 ~ 6Omin, a high success rate of this staining method, double-dye effect, bright color. This method is particularly suitable for medium-sized NADPH-d neurons, and more concentrated brain area for staining. Should pay attention to the application of this method is: ① should minimize the back of immunohistochemical staining; ② 37 ℃ incubation time; Division does not exceed Zh, ③ only