Alfei L, Colombari P T, Cavallo D, Eleuteri P, De Vita R
Animal and Human Biology Department, University of Rome La Sapienza, Italy.
Eur J Histochem. 1993;37(2):183-9.
The suitability of bromodeoxyuridine (BrdU) immunohistochemistry for the study of myosatellite cell proliferation in three subadult carp (Cyprinus carpio) stages (11, 15, 17 cm) was examined. They were injected intraperitoneally with BrdU and fixed one hour late. After fixation and dehydration, white myotomal muscle and small intestine samples were embedded in Histowax. Cross sections mounted on glass slides, were incubated with monoclonal antiBrdU antibody (1:100) after HCl denaturation. After washing twice in PBS, slides were incubated in goat antimouse IgG FITC secondary antibody (1:20). Single cell suspensions were obtained from gut samples. Cellular DNA partially denatured with 2 N HCl, were immunolabelled with monoclonal antibodies against BrdU. Bivariate distribution of BrdU/total cellular DNA content was measured by flow cytometry. Good visualization of BrdU labelled myosatellite cells (4-6%) and small intestine (8-9%) was obtained. Flow cytometric bivariate BrdU/DNA analysis gave evidence of the same proliferative rate in the gut samples. The applicability of these methods to fish tissues further extend the broad range of biological and biomedical investigation in which BrdU immunohistochemistry has been used.
研究了溴脱氧尿苷（BrdU）免疫组织化学在三个亚成体鲤鱼（鲤属鲤鱼）阶段（11、15、17厘米）肌卫星细胞增殖研究中的适用性。对它们进行腹腔注射BrdU，一小时后固定。固定和脱水后，将白色肌节肌和小肠样本包埋在Histowax中。将载玻片上的横截面在盐酸变性后与单克隆抗BrdU抗体（1:100）孵育。在PBS中洗涤两次后，将载玻片在山羊抗小鼠IgG FITC二抗（1:20）中孵育。从肠道样本中获得单细胞悬液。用2N盐酸对细胞DNA进行部分变性，用抗BrdU单克隆抗体进行免疫标记。通过流式细胞术测量BrdU/总细胞DNA含量的双变量分布。获得了BrdU标记的肌卫星细胞（4-6%）和小肠（8-9%）的良好可视化效果。流式细胞术双变量BrdU/DNA分析证明肠道样本中的增殖率相同。这些方法在鱼类组织中的适用性进一步扩展了使用BrdU免疫组织化学的广泛生物学和生物医学研究范围。
