阿尔新蓝染色 (1)
阿尔新蓝、阿尔新蓝-茜素红染色
1、阿尔新蓝染液
100%乙醇80ml
冰醋酸20ml
alcian blue 8 GX 0.1g
充分混匀溶解后，37°静置过夜。
2、无酸双染色剂（阿尔新蓝—茜素红S）
A液：alcian blue 8 GX用于软骨染色。
首先将阿尔新蓝粉末溶解到50%的乙醇中，37°孵育并摇匀，溶解后再加入95%乙醇是浓度达到70%。100ml
A液=5ml 0.4%阿尔新蓝+70ml 95%乙醇+5ml 1M MgCl2 +20ml dH2O 。
B液：0.5% alizarin red S, 0.5g alizarin red S 粉末溶解于100ml蒸馏水中。
混合液中试剂终浓度：0.02% alcian blue，40-60 mM MgCl2,70%乙醇。
在使用前，取10ul的B液和1ml的A液混合使用（现配现用）。
3、斑马鱼整体软骨-骨骼无酸双染色
取各处理组幼鱼，分装于1.5 ml离心管中，每管约50只幼鱼。
1）幼鱼组织固定
麻醉幼鱼，使用4%的中性甲醛缓冲液（由PBS配制）或使用2%PFA固定在1.5ml离心管中，室温振荡10min，放置2h。吸走固定液，并用PBS清洗2次，使用50%乙醇脱水。
2）染色
去除50%乙醇，加入1ml阿尔新蓝-茜素红S染色液到离心管中，质问轻微振荡过夜。
3）漂白
漂白以去掉幼鱼表面色素。漂白液现配现用，由等量的3% H2O2和2%KOH 混合组成，终浓度为1.5%H2O2和1%KOH溶液。染色管中，在去掉染色液后，加入1ml水，倒置混合吸去液体。再新加入1ml漂白液，并将离
心管盖打开，室温放置20min。
4）清理
去掉漂白液，加入20%甘油/0.25KOH室温振荡30min至10h，边振荡边观察胚胎清理状态。然后用50%甘油/0.25%KOH替换之前的清理液，并再次室温振荡2h，放置过夜，显微镜下观察。
5）保存和统计拍照
染色后幼鱼可在50%甘油/0.1%KOH溶液中4°长期保存备用。并在此保存液中利用解剖镜和成像系统拍照。
4、阿尔新蓝软骨染色
1）幼鱼组织固定
麻醉幼鱼，使用1.5ml离心管，固定在静置了两天的4%PFA溶液中，室温过夜。
2）阿尔新蓝染色
吸去PFA后，用95%乙醇溶液洗胚胎2次，彻底吸干后，添加1ml80%乙醇/20%冰醋酸配制的0.1%的阿尔新蓝溶液，取上清染液，染色过夜。
3）洗脱
用60%乙醇/40%PBS溶液冲洗胚胎1次后，换成40%乙醇/60%PBS再冲洗1次，再更换成20%乙醇/80%PBS冲洗一次，最后用PBS缓冲液冲洗2次。
4）清理
吸干PBS溶液后，使用溶解于饱和四硼酸钠溶液中的0.05%trypsin清理胚胎1-3h。
5）漂白
吸去胚胎周围的清理液并用PBS冲洗1次，然后添加1%KOH/3% H2O2溶液侵泡2-4小时。打开离心管盖30°左右静置，并观察胚胎的清理状态。
6）脱色
过1%KOH/3# H2O2：甘油梯度溶液。现用1:3比例，再换成1:1，最后换成3:1（1%KOH/3# H2O2：甘油）
7）保存与拍照
样品保存在50%甘油/50%PBS溶液中或保存在70%甘油中，在此保存液中利用解剖镜和成像系统拍照并统计。
Alcian Blue staining protocol（Development of Cartilage and Bone）
a. Alcian Blue Staining of Cartilage
1. Anesthetize larvae in 5 to 10% methyl ethane sulfonate (i.e., tricaine).
2. Fix in
3.7% neutral buVered formaldehyde (10% formalin in phosphate
buVered saline [PBS]) at room temperature for several hours to overnight.
Care
should be taken not to over ?x the preparation.
3. Wash larvae in PBT (PBT: PBS, 0.1% Tween-20) 3 to 5 times for at least 5
minutes per wash.
4. Transfer into a 0.1% solution of Alcian blue (Sigma, St. Louis, MO) dis-
solved in 80% ethanol and 20% glacial acetic acid for at least 6 to 8 hours to overnight. (Do not overstain). Alcian solution should be syringe ?ltered (0.22 um)
before use.
5. Rinse larvae in ethanol and rehydrate gradually into PBS. Use ethanol, PBT
solutions of 70%-30%, 50%-50%, and 30%-70%, respectively.
6. If ?sh have not been raised in phenyl-thiourea (PTU), remove pigmentation
by bleaching in 3% hydrogen peroxide, 1% potassium hydroxide for 1 to 3 hours.
This reaction should be monitored carefully and stopped once eye pigmentation
b.Microdissection and Mounting of Stained Cartilage
软骨Alcian blue 染色步骤
Fixed nac mutant embryos at 3 and 9 dpf were gently transferred into small 3510 mm Petri dishes with lids using a small plastic transfer pipette and given to students. During the cartilage staining procedure, students wore gloves and goggles and disposed of liquid waste in a designated waste bottle. Without disturbing the embryos in the dish, the acid= ethanol fixative was removed and replaced with the alcian blue cartilage stain consisting of 0.2% w=v alcian blue (Sigma A-3157) diluted in the acid=ethanol fixation solution. The embryos were submerged in the stain for 30 min at room temperature. The alcian blue stain was removed and replaced with the original acid=ethanol fixation solution. The embryos were washed with gentle intermittent plate swirling for at least 10 min to remove any stain not bound to cartilage. After this washing step the acid=ethanol was removed and the embryos were submerged in KOH=glycerol (1% KOH; 50% glycerol) for at least 20 min.