Monoclonal antibodies that label discrete cell types in the mammalian nervous system.
Monoclonal antibodies that label discrete cell types in the mammalian nervous system.
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标记哺乳动物神经系统中离散细胞类型的单克隆抗体。
DOI：
10.1101/sqb.1983.048.01.089
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Horn,JP
中科院分区：
文献类型：
--
作者：
Barnstable,CJ;Akagawa,K;Hofstein,R;Horn,JP
方法单克隆抗体制备。本文（表1）中讨论的大多数抗体的产生已在其他地方进行了描述（Barnstable 1980，1982; Barnstable和Drager 1983; Fekete和Barnstable 1983; R. Hofstein和CJ Barnstable，准备中）。免疫原包括粗制的P2膜沉淀（Whittaker等人，1964）、突触体质膜（Jones和Matus 1974）和扁豆凝集素亲和纯化的糖蛋白（Snary等人，1998）。1977年）。以3周的间隔对小鼠进行至少两次注射200 μ g蛋白质的弗氏佐剂。最后一次注射后三周，对小鼠静脉注射100 μ g蛋白质的等渗盐水溶液。4天后，分离脾细胞并与P3-NSI/1-Ag 4 -1（科勒等人，1976）或P3-X63-Ag 8融合。653（Kearney et al. 1979）浆细胞瘤细胞，使用50%聚乙二醇（Baker 1000）作为融合剂（Galfre et al. 1977; Barnstable 1982）。在HAT培养基中选择杂交培养物（Littlefield 1964），并通过固相放射性结合试验或组织切片免疫细胞化学试验筛选培养物上清液（威廉姆斯1977; Barnstable 1980; Gozes和Barnstable 1982）。通过有限稀释平板接种或在0.3%琼脂中平板接种克隆阳性培养物。使单个克隆适应于在没有HAT的情况下生长，并在组织培养物中大量生长或作为腹水肿瘤生长（Brodsky等人，1979）。免疫细胞化学。给动物注射过量的戊巴比妥，然后用磷酸盐缓冲盐水（PBS）通过心脏灌注，然后用相同缓冲液中的4%多聚甲醛灌注。解剖组织，在相同固定剂中后固定1小时，并浸入30%蔗糖中。然后切下10- 1 J-gin低温恒温器切片并固定在凝胶化载玻片上。将切片与一抗在4 ° C下孵育过夜或在室温下孵育1小时，在PBS中洗涤，并在荧光二抗中在室温下孵育1小时。清洗后，sec-863
METHODSMonoclonal antibody production. The production of most of the antibodies discussed in this paper (Table 1) has been described elsewhere (Barnstable 1980, 1982; Barnstable and Drager 1983; Fekete and Barnstable 1983; R. Hofstein and CJ Barnstable, in prep.). Immunogens have included crude P2 membrane pellets (Whittaker et al. 1964), synaptosomal plasma membranes (Jones and Matus 1974), and lentil lectin affinity-purified glycoproteins (Snary et ai. 1977). Mice were given at least two injections of 200 gg of protein in Freund's adjuvants at 3-week intervals. Three weeks after the final injection, mice were given an intravenous injection of 100 gg of protein in isotonic saline. Four days later, spleen cells were isolated and fused with either P3-NSI/1-Ag4-1 (Kohler et al. 1976) or P3-X63-Ag8. 653 (Kearney et al. 1979) plasmacytoma cells using 50% polyethylene glycol (Baker 1000) as fusing agent (Galfre et al. 1977; Barnstable 1982). Hybrid cultures were selected in HAT medium (Littlefield 1964), and culture supernatants were screened either by a solid-phase radioactive binding assay or by an immunocytochemical assay on tissue sections (Williams 1977; Barnstable 1980; Gozes and Barnstable 1982). Positive cultures were cloned either by plating at limiting dilution or by plating in 0.3% agar. Individual clones were adapted to growth without HAT and grown in bulk in tissue culture or as ascitic tumors (Brodsky et al. 1979). lmmunocytochemistry. Animals were injected with an overdose of Nembutal and then perfused through the heart with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in the same buffer. Tissues were dissected out, postfixed for 1 hour in the same fixative, and sunk in 30% sucrose. Then 10-1J-gin cryostat sections were cut and mounted on gelatinized slides. Sections were incubated with primary antibody either overnight at 4~ or for 1 hour at room temperature, washed in PBS, and incubated in fluorescent second antibody for I hour at room temperature. After washing, sec-863
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DOI：
--
发表时间：
1949
期刊：
影响因子：
--
作者：
M. A. Bouman
通讯作者：
M. A. Bouman