Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor.
Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor.
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DOI：
10.1083/jcb.56.3.838
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发表时间：
1973-03
期刊：
影响因子：
--
通讯作者：
Leblond CP
中科院分区：
文献类型：
--
作者：
Weinstock M;Leblond CP
材料与方法体重30~40g的SD大鼠静脉注射[GAP]磷酸(2.5mCI/只，无载体)、L-[~3H]丝氨酸(2.5mCI/只，SP act 2.23Ci/mmo1)或L-[2，3-~3HPro(2.5mCi/只，SP act 45.7ci/mmol)]。在乙醚麻醉下，分别于注射后5、10、20、30、90nfin，以及注射后4、30h，每隔一定时间处死动物2只。注射[H]丝氨酸后，每隔30、90、4h各处死1只动物，固定剂为2.5%戊二醛加入0.05M S6rensen‘s磷酸盐缓冲液中，外加0.1%蔗糖和0.5%葡萄糖。在使用L-[2，~-Gh]脯氨酸的实验中，使用的固定剂是3%甲醛(英国雷丁埃默格林TAAB实验室)，在0.1M S6rensen‘s磷酸盐缓冲液中加入0.1%蔗糖。两种固定剂的最终pH值均为7.2~7.3。在室温下灌流15min后，上颌切牙在新鲜固定剂中浸泡2~3h，然后在4℃(10℃)下用EDTA脱矿2wk。尽管2wk的时间足以彻底脱矿，但在用[3~P]磷酸盐进行的实验中，一些牙齿被脱矿3或4wk。标本在0.15M的S6rensen‘s缓冲液中清洗过夜，用剃须刀片横向切片成1 mm厚的切片，在1%OsO4的0.1IV中后处理1-2小时。
MATERIALS AND METHODSSherman rats weighing 30-40 g received an intravenous injection of either [gaP] phosphate (2.5 mCi per animal; carrier-free), L-[3H] serine (2.5 mCi per animal; sp act 2.23 Ci/mmol) or L-[2, 3-3H] proline (2.5 mCi per animal; sp act 45.7 Ci/mmol). Two animals per time interval for each precursor were sacrificed under ether anesthesia by perfusion of fixative through the left ventricle 5, 10, 20, 30, and 90 nfin, and 4 and 30 h after injection. After the injection of [~ H] serine, however, one animal per time interval was sacrificed after 30 and 90 rain, and 4 h. The fixative consisted of 2.5% glutaraldehyde in 0.05 M S6rensen's phosphate buffer with the addition of 0.1% sucrose and 0.5% dextrose. In the experiments using L-[2,~-gH] proline, the fixative employed was 3% formaldehyde (TAAB Laboratories, Emmer Green, Reading, England) in 0.1 M S6rensen's phosphate buffer with 0.1% sucrose added. The final pH of either fixative was 7.2-7.3. After perfusion for 15 min at room temperature the maxillary incisor teeth were immersed in fresh fixative for 2-3 h and demineralized in EDTA for 2 wk at 4 C (10). Although a 2-wk period is sufficient for exhaustive demineralization, some teeth were denfineralized for 3 or 4 wk in the experiments conducted with [3~ P] phosphate. Specimens were washed overnight in 0.15 M S6rensen's buffer, sliced transversely with razor blades into 1-mm thick sections, postfixed for 1-2 h in 1% OsO4 in 0.1 IV [S6rensen's buffer, dehy-