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运用定量分析方法对NADPH－d组化染色方法进行了初步探讨。实验对象采用在鼠海马，固定后的标本冰冻冠状连续切片35urn，切片染色封装后，光镜下图像分析方法定量观测染色效果。结果显示以下几个因素对染色的影响颇大。1．染液的配方：我们推荐NBT浓度为0.1mg／ml，NADPH－d浓度为1mg／ml，0．IMPBSpH7．3，Triton－100浓度为03％的配方。提高NBT浓度至0．2、04、0sing／ml，染色速度加快，但易造成深染细胞的过染而使神经元形态不清并易造成切片污染。NADPH—d的浓度最小值是ling／ml，较低的浓度05～08mg／ml染色速度减慢且着色浅淡，染色细胞数量减少。较高的浓度1．2、1．sing／ml与ling／ml的染色效果类似，但染色时间缩短。2．固定液：4％多聚甲醛固定的标本中，NADPH－d染色阳性神经元见于海马除锥体细胞层的其它各层，染色细胞为原生型NOS神经元，4％多聚甲醛＋1．25％戊H醛固定的标本可使锥体细胞显示淡阳性，似可以使内皮型NOS神经元（eNOS）显示阳性。因此，选用不同固定液有助于确定NOS的类型。3．染色时间：37C下温育以40～60分钟为宜，时间过长可造成某些胶质细胞的染色并可使染色过深。通过试验我们认为：对脑组织进行NADPH－d染色的适宜条件为：NBT浓度：0．l一0．12ms／ml，NADPH－d为
Quantitative analysis of NADPH-d histochemical staining method was initially explored. The experimental objects were frozen in the rat hippocampus, and the frozen specimens were fixed in 35urn sections. The sections were stained and encapsulated, and the images were analyzed quantitatively under a light microscope. The results show that the following factors have a significant impact on the staining. 1. Dye formula: We recommend NBT concentration of 0.1mg / ml, NADPH-d concentration of 1mg / ml, IMPBS pH7.3, Triton-100 concentration of 03% of the formula. NBT concentration increased to 0.2,04,0sing / ml, faster staining, but easily lead to stained cells stained deep neuronal morphology and easy to cause section pollution. The minimum concentration of NADPH-d is ling / ml, the lower the concentration of 05 ~ 08mg / ml slows down and coloration pale, stained cells decreased. The higher concentration of 1.2,1. The staining effect of sing / ml and ling / ml is similar, but the staining time is shortened. 2. Fixation fluid: NADPH-d-stained neurons were found in other layers of hippocampus except pyramidal cells in 4% paraformaldehyde-fixed specimens. The stained cells were native NOS neurons, 4% paraformaldehyde + 1.25% Hyaluronan-fixed specimens showed pyramidal cells showed a positivity of positive, it seems that the endothelial NOS neurons (eNOS) showed positive. Therefore, the choice of different fixative helps to determine the type of NOS. 3. Dyeing time: 37C under the incubation of 40 to 60 minutes is appropriate, too long can cause some glial cells staining and dyeing too deep. Through the experiment we think: suitable conditions for NADPH-d staining of brain tissue: NBT concentration: l a 0.12ms / ml, NADPH-d is