CN110646270B - 一种适于翘嘴鲌幼体骨骼染色的方法 - Google Patents
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Abstract
一种适于翘嘴鲌幼体骨骼染色的方法,具体包括以下步骤:步骤一:用4%PFA溶液将幼体翘嘴鲌样本固定;步骤二:用不同浓度梯度的乙醇溶液将幼体翘嘴鲌样本复水,每次时间10~25min;步骤三:将复水后的标本用双氧水溶液漂白,漂白时间为28~30min;步骤四:将漂白后的标本用DEPC溶液浸泡25~35min;步骤五:用1%KOH溶液透明标本,时间为18~25min;步骤六:更换新的1%KOH溶液中,然后加入适量的茜素红染液,染色时间为60~75min;步骤七:将染色后的标本用新的1%KOH溶液进行退色;步骤八:将退色后的标本用50%甘油进行处理;步骤九:将标本放置在100%甘油中保存。此方法根据幼体翘嘴鲌的特性制定的方法简单易行、实验试剂及仪器简单,可获得身体透明、肌间骨有效染色且清晰的翘嘴鲌幼体标本。
Description
技术领域
本发明属于骨骼染色技术领域,具体涉及一种适于翘嘴鲌幼体骨骼染色的方法。
背景技术
翘嘴鲌(Culter alburnus Basilewsky)肉质洁白、鲜嫩,营养价值高,生长速度快,已成为中国特别是长江三角洲地区的主要名特淡水养殖品种。与我国其他淡水主养鱼类,如草鱼、鲢鱼、鳙鱼、鲤鱼、鲫鱼、鲂鱼、乌鳢等一样,翘嘴鲌大多有肌间刺,不仅达不到在国际市场上优质鱼类的品质要求,还严重影响其食用及商品品质,因此培育无肌间刺的翘嘴鲌品种有着巨大的经济价值和市场。
鱼的肌间刺(intermuscular bone)是分布于鱼类肌间隔中的硬骨小刺,也称为肌间小骨,当前国内外关于鱼类肌间刺的相关研究基本停留于形态、分布等描述阶段以及对不同鱼类中肌间刺类型的比较分析,至于肌间刺在体内形成的分子机制以及相关的发育遗传调控均未见报道;肌间刺发育调控基因的确定首先必须清楚肌间骨出现的时序及骨化模式,对翘嘴鲌幼体进行整体骨骼染色可清晰地观察到翘嘴鲌肌间骨的骨化模式及出现时间、肌间骨不同发育阶段的确定及材料选取,并结合二代高通量测序技术,有助于我们快速筛选到肌间骨发育调控基因。
授权号为CN105532642B的文件公开了一种团头鲂成鱼肌间骨染色的方法及应用,得到的标本可清楚观察团头鲂成鱼肌间骨的附着位置及形态,但是该方法不能得到品质较高的翘嘴鲌幼体骨骼染色的标本。
发明内容
针对上述存在的问题,本发明的目的是提供一种适于翘嘴鲌幼体骨骼染色的方法,针对幼体的发育状况进行骨骼染色的工艺进行合理的优化,得到的幼体骨骼标本可清晰地观察翘嘴鲌幼体肌间骨的附着位置及形态,工艺简单,易于操作。
为了实现上述目的,本发明的技术方案如下:
一种适于翘嘴鲌幼体骨骼的染色方法,包括以下步骤:
步骤一:用4%PFA溶液将幼体翘嘴鲌样本固定0.5~1天;
步骤二:用不同浓度梯度的乙醇溶液将幼体翘嘴鲌样本复水,每次时间10~25min;
步骤三:将复水后的标本用双氧水溶液漂白,漂白时间为28~30min;
步骤四:将漂白后的标本用DEPC溶液浸泡25~35min;
步骤五:用1%KOH溶液透明标本,时间为18~25min;
步骤六:更换新的1%KOH溶液中,然后加入适量的茜素红染液,染色时间为60~75min;
步骤七:将染色后的标本用新的1%KOH溶液进行退色;
步骤八:将退色后的标本用50%甘油进行退色和缩水处理;
步骤九:将标本放置在100%甘油中保存。
优选地,所述幼体翘嘴鲌为10~30天间的多个生长阶段的幼体。
在本方案设计中,10~30天间的幼体翘嘴鲌骨骼生长发育迅速,其活动和体质状况明显增加,其内的调控基因活跃量大且大部分为蛋白质,因此采用低浓度的缓冲PFA溶液进行固定,不会对幼体标本进行损伤,固定性好且穿透力强,因此本方案中采用4%PFA溶液是最适于幼体翘嘴鲌的固定液。
在本方案设计中,考虑到目的是为了骨骼清晰可见,使用具有漂白效果的双氧水对幼鱼的组织进行漂白,可减少后续处理步骤中透明所需的时间,并且透明效果更佳,更有利于染色剂的染色,双氧水对于体积较大的标本的漂白效果不佳,但对于体积较小的幼鱼来说能进行有效漂白处理,且双氧水在后续处理中易去除,不会对后续步骤产生不良影响。
在本方案设计中,乙醇溶液的浓度梯度为:75%乙醇、50%乙醇、30%乙醇。
在上述步骤中,相关试剂的配制具体为:4%PFA:100mL1×PBS+4g多聚甲醛粉末;75%乙醇:75mL无水乙醇+5mL双蒸水;50%乙醇:50mL无水乙醇+50mL双蒸水;30%乙醇:30mL无水乙醇+70mL双蒸水;1%双氧水:10mL30%双氧水+290mL双蒸水;1% KOH溶液:1g氢氧化钾+100mL双蒸水;茜素红染液:1g茜素红粉末+100mL1% KOH溶液(染色相关试剂可等比例扩大配制);50%甘油:100%甘油与1% KOH溶液1:1混合;100%甘油内加入有少许具有杀菌和抑菌效果的麝香草酚。
优选地,在复水、漂白、浸泡、透明处理过程中均不移动标本。
在本方案设计中,由于幼体翘嘴鲌较小,不宜移动,而本方案的处理步骤皆可满足标本不移动的需要,最后得到的标本完成性好。
一种适于翘嘴鲌幼体肌间骨的观察方法,将上述的翘嘴鲌标本肌间骨段进行观察,确定不同生长时期的翘嘴鲌幼体肌间骨的骨化模式及确定翘嘴鲌肌间骨的出现时间,从而为进一步筛选肌间骨发育调控基因奠定了基础。
综上所述,本发明具有以下有益效果:
本方案根据幼体翘嘴鲌的特性制定的方法简单易行、实验试剂及仪器简单,可获得身体透明、肌间骨有效染色且清晰的翘嘴鲌幼体标本,可清楚地观察到肌间骨的附着位置及形态,为进一步研究翘嘴鲌肌间骨的机制奠定了基础。
附图说明
附图1为本发明孵出后10天幼体翘嘴鲌的骨骼染色图。
附图2为本发明孵出后20天幼体翘嘴鲌的骨骼染色图。
附图3为本发明孵出后30天幼体翘嘴鲌的骨骼染色图。
具体实施方式
实施方法
从孵出后10天后每隔10天采集幼体翘嘴鲌,每次10尾,进行骨骼染色,具体步骤如下:
(1)将幼体翘嘴鲌麻醉后,去除内脏,固定在4%PFA中1天(固定时若溶液变浑浊,需要替换新的4%PFA溶液,并且清除标本体表脱落的杂质),依次经过75%乙醇、50%乙醇、30%乙醇梯度复水,每次15min,最后用双蒸水漂洗两次,每次10min;
(2)将幼体翘嘴鲌用1%双氧水漂白30min,然后用足量的DEPC水浸泡30min,再用1%KOH溶液透明20min;
(3)更换新的1%KOH溶液,加入适量的茜素红染液(添加量比例为5:1)进行染色,染色1h后更换新的1%KOH溶液对标本进行浸泡,褪去标本表面的染色液(褪色期间可根据褪色程度更换1%KOH溶液);
(4)将标本置于50%甘油中,直至体表的染色褪去至骨骼清晰可见(褪色时间一般为24~60h,具体时间根据褪色程度观察而得);
(5)最后将标本置于加入少许麝香草酚的100%甘油(添加量为0.05%)内进行保存。
上述步骤中相关试剂的配制具体为:
4%PFA:100mL1×PBS+4g多聚甲醛粉末;
75%乙醇:75mL无水乙醇+5mL双蒸水;
50%乙醇:50mL无水乙醇+50mL双蒸水;
30%乙醇:30mL无水乙醇+70mL双蒸水;
1%双氧水:10mL30%双氧水+290mL双蒸水;
1% KOH溶液:1g氢氧化钾+100mL双蒸水;
茜素红染液:1g茜素红粉末+100mL1% KOH溶液;
50%甘油:100%甘油与1% KOH溶液1:1混合(1×PBS、多聚甲醛粉末、无水乙醇、30%双氧水、氢氧化钾、茜素红粉末、100%甘油均购于生物公司)。
实施例1
本实施例采集孵出后10天的10尾幼体翘嘴鲌根据上述步骤进行骨骼染色,观察选出表现较好的标本如附图1所示。
实施例2
本实施例采集孵出后20天的10尾幼体翘嘴鲌根据上述步骤进行骨骼染色,观察选出表现较好的标本如附图2所示。
实施例3
本实施例采集孵出后30天的10尾幼体翘嘴鲌根据上述步骤进行骨骼染色,观察选出表现较好的标本如附图3所示。
观察比较附图1~3所呈现的不同时期的肌间骨发育特征,10天时,肌间骨未出现,但中轴骨发育完整;20天时,一些小肌间骨陆续出现;30天时,肌间骨基本发育成熟;因此可初步确定翘嘴鲌肌间骨出现的时间在孵化后20天左右。
本文中所描述的具体实施例仅仅是对本发明精神作举例说明。本发明所属技术领域的技术人员可以对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代,但并不会偏离本发明的精神或者超越所附权利要求书所定义的范围。
Claims (7)
1.一种适于翘嘴鲌幼体骨骼染色的方法,其特征在于,包括以下步骤:
步骤一:用4%PFA溶液将幼体翘嘴鲌样本固定;
步骤二:用不同浓度梯度的乙醇溶液将幼体翘嘴鲌样本复水;
步骤三:将复水后的标本用双氧水溶液漂白;
步骤四:将漂白后的标本用DEPC溶液浸泡;
步骤五:用1%KOH溶液透明标本;
步骤六:更换新的1%KOH溶液中,然后加入适量的茜素红染液进行染色;
步骤七:将染色后的标本用新的1%KOH溶液进行退色;
步骤八:将退色后的标本用50%甘油进行处理;
步骤九:将标本放置在100%甘油中保存;
所述步骤二~九的处理过程中幼体翘嘴鲌标本均不移动。
2.根据权利要求1所述的一种适于翘嘴鲌幼体骨骼染色的方法,其特征在于,所述幼体翘嘴鲌为10~30天间的多个生长阶段的幼体。
3.根据权利要求1所述的一种适于翘嘴鲌幼体骨骼染色的方法,其特征在于,所述步骤二中乙醇溶液的浓度梯度为:75%乙醇、50%乙醇、30%乙醇。
4.根据权利要求1所述的一种适于翘嘴鲌幼体骨骼染色的方法,其特征在于,所述50%甘油配比为:100%甘油:1% KOH溶液=1:1。
5.根据权利要求4所述的一种适于翘嘴鲌幼体骨骼染色的方法,其特征在于,所述1%KOH溶液配比为:氢氧化钾:双蒸水=1g:100ml。
6.根据权利要求1所述的一种适于翘嘴鲌幼体骨骼染色的方法,其特征在于,所述4%PFA溶液配比为:100mL1×PBS+4g多聚甲醛。
7.一种基于权利要求1~6任一项方法 制备的翘嘴鲌幼体骨骼标本的观察方法,其特征在于,观察对比多个生长阶段的翘嘴鲌幼体骨骼标本,确定各个翘嘴鲌幼体骨骼标本肌间骨的骨化模式及肌间骨的出现时间。
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