英文版

Whole mount in situ hybridization of zebrafish

 

 

Day 1

Embryo preparation. Place embryo collector into the tank in the evening.   

 

Day 2

Collect the embryo in the morning. Embryos are washed and cleaned.  .

Fix the embryos at expected stages.

 

Fixation

Fixative: 4% paraformaldehyde (in 1×PBS：130 mM NaCl, 7 mM Na2HPO4.2H2O, 3 mM NaH2PO4)

10×PBS stock solution: mix 76 g NaCl, 9.9 g Na2HPO4, 3.6g NaH2PO4. Dissolve them in less than 1 liter nanopure water, adjust to pH7.0 and a final volume of 1 liter. Autoclave for 35 minutes to sterilize.

130 mM NaCl = 10 ×0.13 X 58.44=76 g NaCl

7 mM Na2HPO4 = 10× 0.007×142 = 9.9 g Na2HPO4

3 mM NaH2PO4 = 10× 0.003×120 = 3.6 g NaH2PO4

Paraformaldehyde (PFA) (Mallinckrodt 2621).

Weigh 2 g PFA and dissolve it in 50 ml 1×PBS. (PFA is hard to dissolve.) Incubate the mixture at 65oC for around 2 hours. Shake the mixture during the incubation). After dissolved, the solution is stored at 4oC. Use it within 3 days (72 hours).

 

Drain water as possible as you can. Add fixative directly to the 1.5 ml eppendorf microcentrifuge tube.  Replace the solution with fresh fixative. Repeat 3 or 4 times. Finally, end up with 100% fixative.  

 

Generally, the fixed embryos will place at 4oC overnight to allow the fixation process completely.  (〈24hours)

 

Dehydration

Dehydrate embryos according to age as follows:

       1 cell to 80% epiboly :

                    step 1: 25% methanol/75% fixative for 5 minutes at room temperature

                    step 2: 50% methanol/50% fixative for 5 minutes at room temperature

                    step 3: 100% methanol for 5 minutes at room temperature

            For doing step 1, aspirate 1/4 of the fixative, add the same amount methanol into the tube; for step 2, take out half of the solution in the tube, add the same amount methanol into the tube; for step 3, remove most of the solution out of the tube, add fresh methanol, repeat 2 times and then end up with 100% methanol.

            Be very careful to hand the embryos, the early stages (up to balstula) of embryos are very fragile.

     12 hours and older embryos:

                    step 1: 50% methanol/50% fixative for 5 minutes at room temperature

                    step 2: 100% methanol for 5 minutes at room temperature

 

Clearance

Store the embryos at -20oC at least 1 hour before use. At this step, embryos may be safe forever.(Several months is definitely OK.)

 

Day 3

Rehydration

Wash embryos according to age as follows:

       1 cell to 80% epiboly :

                    step 1: 25% PBST/75% methanol for 5 minutes at room temperature

                    step 2: 50% PBST/50% methanol for 5 minutes at room temperature

                    step 3: 100% PBST for 5 minutes at room temperature, twice

            For doing step 1, aspirate 1/4 of the methanol, add the same amount PBST into the tube; for step 2, take out half of the solution in the tube, add the same amount PBST into the tube; for step 3, remove most of the solution out of the tube, add fresh PBST, repeat one more time.

            Be very careful to hand the embryos, the early stages (up to balstula) of embryos are very fragile.

     12 hours and older embryos:

                    step 1: 50% PBST/50% methanol for 5 minutes at room temperature

                    step 2: 100% PBST for 5 minutes at room temperature, twice

PBST: 1×PBST plus 0.1% Tween-20 (Sigma P-9416) (1 ul Tween-20 per ml 1×PBS)

 

Proteinase K treatment

Remove the washing PBST, add new fresh PBST to 1.5 ml, add the desired proteinase K directly into the tube and gently roll the tube to increase the proteinase K diffusion. (Vortex the tube and make sure the precipitate be mixed evenly before use).

According to age as follows:

       1 cell to 20 hours: not  treat.

    > 24 hours: 10 ug/ml for 7-8 minutes at room temperature

      old than 36 hours: 10 ug/ml for 10-12 minutes at room temperature

           Proteinase K (Sigma P-2308); Stock solution (10 mg/ml = 10 ug/ul): weigh 10 mg proteinase K and dissolve in 1 ml nanopure water. Aliquot 50 ul each and store at -20oC. Thaw at room temperature before usage. Long storage may appear to see white precipitate after thawing. Vortex the tube and make sure the precipitate be mixed evenly.

 

After digestion:

       take out the solution, rinse the embryos with PBST, then wash with fresh PBST for 5 minutes at room temperature.

 

Postfixation

Remove PBST, add the fixative (4% PFA, warm to room temperature) to the tube, remove the PFA, then add fresh one. Fix the embryos for 20 minutes at room temperature

wash with PBST twice for 5 minutes at room temperature

 

 

Hybridization

using 1.5 ml microcentrifuge tube

Remove PBST, add hyb- to pre-hybridize embryos for 5 minutes at 55oC (50-65oC)

Remove the hyb-, add hyb+ and incubate for 4 hours (up to 6 hours) at 55oC

 

20×SSC

      For 1 liter: 1×3 mol = 3 × 58.44 =175.3 g NaCl

                  1× 0.3 mol = 0.3 × 294.1 = 88.2 g Na₃.Citrate.2H₂0

      adjust pH=7 using HCl. autoclave 35 minutes for sterilizeization.

hyb-: 50% formamide, 5XSSC, 0.1% Tween-20 (0.5 formamide, 0.25(1/4) 20×SSC, 0.25(1/4) H2O, 1 ul of Tween per ml of the solution).

hyb+: hyb-, 500 ug/ml yeast RNA, 50 ug/ml heparin.

Formamide (Fisher BP228-100, Superpure, been treated with mixed resin--deionized)

Yeast RNA (Sigma R6750): dissolve 50 mg RNA in 1 ml nanopure water (50 mg/ml); store at -20oC Þ 10 ul per ml of hyb-

Heparin (Sigma H3393): dissolve 50 mg heparin in 1 ml nanopure water (50 mg/ml); store at -20oC Þ 1 ul per ml of hyb-

 

add probes to 1 ng/ul to fresh hyb+, heat at 65oC for 10 minutes, chill it on ice for 5 minutes, then remove the prehybridization hyb+ and add the fresh hyb+ containing the probe (Dig-labeled).30-40 embryo/100ul hyb+

 

Day 4

 

Probe Removal

probes can be reused in a limited time when store at -20oC. Results show if store more then 2 months, the probes reused gave very high background, but reused it within one week gave very good results.

 

Washes: all steps at 60oC (60oC-65oC)

     2 times for 30 minutes at 60oC in 2×SSCT, 50% formamide (0.5 formaimide, 0.1 20×SSC, (0.1%) 1 ul of Tween per ml of the solution)

     1 time for 15 minutes in 2×SSCT

     2 times for 30 minutes in 0.2XSSCT(2XSSCT/H2O  1/9)

Pre-block washes at room temperature

     3 times with MABT for 5 minutes

     MAB X 2 liters pH7.5

     100 mM Maleic acid (Sigma M-0375) = 23.2 g (2 × 0.1 ×116.1)

     150 mM NaCl = 17.5g (2 × 0.15 × 58.44)

     add solid (? g) to adjust pH=7.5, autoclave for 35 minutes to sterlize.

 

Block

     block for 1 hour ( up to 4 hour) with 2% blocking regent, 10% sheep serum and 70% MAB. Add about 1 ml per 1.5 ml tube.

        Blocking regent (1096 176, Boehringer Mannheim, now Roche?). 10% stock solution. Weigh 10 g blocking regent and dissolve it in 100 ml MAB, shaking and heating in a microwave oven for 1 minutes (?), autoclave for 25 minutes and then aliquot into 14 ml and store at -20oC.  

       Sheep serum (Sigma: S2263), aliquot and store at -20oC

 

Immnohybridization

        add fresh blocking solution with 1:2500 (1:1000 ~ 1:5000) dilution of alkaline posphatase conjugated digoxigenin antibody (Anti-Digoxigenin-AP Fab fragment); incubate overnight at 4oC (12 ~ 20 hours). Add 350 ~ 450 ul per tube which containg 0.03 ~ 0.04 ml embryos (1/3 of 0.1 ml embryos).

        Anti-Digoxigenin-AP Fab fragment (from sheep) (1093274, Boehringer Mannheim, now Roche?) store at 4 oC, good for longer than 1 and half year: since 1/19/99)

 

Day 5 Antibody removal

 

Washes

Wash with MABT at room temperature with gently shaking. Change solution every 45 ~ 60 minutes, let it go overnight (12 ~ 18 hours) at room temperature . Totally, solutions must be changed at least 8 times.

Antibody may be saved at 4 oC and reused (not try yet)

 

Day 6 Visualization

Change the overnight MABT washing solution with fresh MABT last time, start to prepare equilibration buffer.

Change MABT with the equilibration containing 0.5 mg/ml of levamisol (Sigma L-9756) and incubate for 10 minutes. And at the time, transfer the embryos to 24-well tissue culture plate (Falcon 3047, Multiwell tissue culture plate).

Drain the equilibration buffer. Add new equilibration buffer containing the substrate NBT/BCIP (4.5/3.5ul/ml) .solution (Boehringer Mannheim, 1681 451) and 1.2 mg/ml of levamisol. Wrap the plate with foil and incubate the embryos at room temperature for 6 hours (up to 7 hours). If satin for 12 hours (up to 48 hours), there is still no background in the sense control experiments, but the yolk become redish and not good for picturing (when photographing, yolk appears black, no longer transparent though you may see it is still good when observing under microscope)

 

   Levamisol (Sigma L-9756): make a 0.2 mg/ul solution eg, weigh 20 mg and dissolve in 100 ul nanopure H2O. Store at 4 oC and use it within 7 days.

   NBT/BCIP stock solution (1681 451, Boehringer Mannheim, now Roche?): add 100 ul of the stocking solution to 5 ml the equilibration buffer. Or,

4.5 ul of 75 mg/ml NBT (1 087 479, Boehringer Mannheim, now Roche?) in 70% dimethylformamide (Mallinckrodt 4929) and 3.5 ul of 50 mg/ml BCIP (760 994, Boehringer Mannheim, now Roche?) in 100% dimethylformamide are addedd to 1 ml of the equilibration buffer.

equilibration buffer: 1ml 1M NaCl +1ml 1M Tris-Hcl PH9.5+1ml 0.5M MgCl2+7ml H2O+0.1% TWEEN-20

中文版（翻译）

 

第一天

胚胎准备。在晚上将胚胎收集器放入水箱。

第二天

早上收集胚胎。胚胎是清洗和清洁　解决胚胎在预期的阶段。

固定

固定剂:4%多聚甲醛(1×PBS:130毫米氯化钠,Na2HPO4.2H2O 7毫米,3毫米NaH2PO4)

　　10×PBS股票的解决方案:76克氯化钠混合,9.9 g Na2HPO4,3.6 g NaH2PO4。溶解在不到1升nanopure水,适应pH7.0和最终体积的1升。高压蒸汽消毒35分钟。

　　130毫米氯化钠= 10×0.13 X 58.44 = 76克氯化钠

　　7毫米Na2HPO4 = 10×0.007×142 = 9.9 g Na2HPO4

　　3毫米NaH2PO4 = 10×0.003×120 = 3.6 g NaH2PO4

　　多聚甲醛(PFA)(Mallinckrodt 2621)。

　　重量2 g PFA和溶解在50毫升1×PBS。(PFA很难溶解。)孵化混合物在65 oc 2小时左右。孵化期间摇混合物)。溶解后,解决方案是储存在4摄氏度。3天(72小时)内使用它。

　　

　　废水尽可能。固定剂直接添加到1.5毫升埃普多夫微型离心机管。用新鲜的固定剂替代解决方案。重复3或4次。最后,最终以100%的固定剂。

　　

一般来说,固定胚胎在一夜之间将在4摄氏度,允许固定过程完整模型

 

脱水胚胎根据年龄如下:

　　1细胞80%外包:

　　步骤1:25%甲醇/ 75%固定剂在室温下5分钟

　　步骤2:50%甲醇/ 50%固定剂在室温下5分钟

　　步骤3:100%甲醇在室温下5分钟

　　进行步骤1,吸入1/4的固定剂,添加相同量甲醇进入管;第二步,拿出一半的解决方案在管,添加相同量甲醇进入管;步骤3,去除大部分的解决方案的管,添加新鲜甲醇,重复2次,然后最终以100%甲醇。

　　小心把胚胎,胚胎的早期阶段(balstula)是非常脆弱的。

胚胎12小时及以上:

　　步骤1:50%甲醇/ 50%固定剂在室温下5分钟

　　步骤2:100%甲醇在室温下5分钟

　　

　　间隙

　　存储胚胎在-20 oc使用前至少1小时。在这一步中,胚胎可能永远是安全的。(几个月绝对是好的。)

第三天

再水化，再水合

 

按年龄清洗胚胎如下：

 

1个细胞至80%表观：

 

第一步：25%PBST/75%甲醇，室温下5分钟。

 

步骤2：50%PBST/50%甲醇在室温下作用5分钟

 

步骤3：室温下100%PBST 5分钟，两次

 

对于步骤1，吸出1/4的甲醇，将相同量的pbst加入到管道中；在步骤2中，取出一半的溶液，在管道中加入相同量的pbst；对于步骤3，reMO。 把大部分溶液从管子里拿出来，加入新鲜的PBST，再重复一次。

 

小心的处理胚胎，胚胎的早期阶段(到压载期)是非常脆弱的。

 

12小时及以上胚胎：

 

步骤1：50%PBST/50%甲醇在室温下5分钟

 

步骤2：100% PBST在室温下5分钟，两次

 

PBST：1×PBST+0.1%吐温-20(西格玛P-9416)(1μl吐温-20/ml 1×pbs)

 

蛋白酶K处理

 

取下洗涤后的PBST，加入新的新鲜PBST至1.5ml，将所需蛋白酶K直接加入到试管中，轻轻摇动试管，以增加蛋白酶K的扩散。

（涡流管并使S 在使用前，将沉淀物均匀混合。

 

按年龄分列如下：

 

1细胞至20小时：不治疗。

 

>24小时：室温下10微克/毫升，持续7-8分钟。

 

>36小时：室温下10微克/毫升，10-12分钟。

 

蛋白酶K(Sigma P-2308)；原液(10 mg/ml=10μg/ul)：称重10 mg蛋白酶K，溶解于1ml纳米水中。每箱50升，以摄氏-20度储存。在室温下解冻 使用。长时间的储存可能会在解冻后出现白色沉淀。旋转管道，确保沉淀物均匀混合。

 

售后：消解

 

取出溶液，用PBST冲洗胚胎，用新鲜的PBST在室温下洗5分钟。

 

posterization多色调分色法

 

取出PBST，加入固定剂(4%PFA，室温加热)到管子上，取出PFA，再加入新鲜的PFA。室温下将胚胎固定20分钟。

 

室温下用PBST洗两次5分钟。

 

杂交

 

使用1.5毫升离心管

 

去除pbst，在55 oC(50-65 oC)下，加入Hyb-到预杂交胚胎5分钟。

 

取出Hyb-，加入Hyb，在55℃孵育4小时(最多6小时)。

 

20×Sv

 

For 1 liter：1×3ml = 3×58.44 = 175.3g NaCl

 

1×0.3 mml = 0.3×254.1＝88.2g Na3 . Citratt.2H20

 

用高压釜35分钟调节pH=7进行消毒。

 

Hyb-：50%甲酰胺，5 XSSC，0.1%吐温-20(0.5甲酰胺，0.25(1/4)20×SSC，0.25(1/4)H2O，1μl吐温/ml溶液)。

 

Hyb-，500μg/ml酵母RNA，50μg/ml肝素。

 

甲酰胺(Fisher BP228-100，超纯，用混合树脂-去离子处理)

 

酵母RNA(Sigma R 6750)：将50毫克RNA溶于1毫升纳米水(50毫克/毫升)；储存于-20 oC(每毫升海布10微升)。

 

肝素(Sigma H 3393)：将50毫克肝素溶解于1毫升纳米水(50毫克/毫升)；储存于-20 oC(每毫升海布1微升)。

 

将探针加入1 ng/L的新鲜HYB+中，在65℃下加热10分钟，在冰上冷却5分钟，然后除去预杂交HYB+，并加入含有探针的新鲜HYB+（GED标记）。 胚胎/100 ul Hyb

第四天

 

探针去除

 

探针可以在有限的时间内重复使用，当存储在-20摄氏度时。结果表明，如果储存时间超过2个月，重复使用的探针本底很高，但在一周内重复使用，效果很好。 .

 

洗涤：所有步骤在摄氏60度(摄氏60度至摄氏65度)

 

2次，60℃，2×SSCT，50%甲酰胺(0.5福尔马胺，0.1 20×SSC，0.1%)每毫升吐温1μl，每次30 min。

 

1次，15分钟，2×SSCT

 

0.2XSSCT(2 XSSCT/H2O 1/9)2次30分钟

 

室温预块洗涤

 

3次使用MABT 5分钟

 

MAb X2升pH7.5

 

100mm Maleic acid Sigma M-0375) = 23.2 g / L 2 × 0.1 × 116.1)

 

150mM NaCl = 17.5g（2×0.15×58.44）

 

加入固体(？g)调节pH=7.5，高压釜35分钟进行杀菌。

 

布洛克，赫伯特·劳伦斯（（生于 1909） 美国卡通片制作者，优秀作品曾见诸<华盛顿邮报> 及遍布全国的200多家报纸。他获得了1942年和1954年的普利策奖）

 

用2%封闭液、10%羊血清和70%MAB阻断1小时(至4h)。每1.5毫升管加约1毫升。

 

阻挡摄政者(1096 176年，博林格曼海姆，现在罗氏？)10%的库存溶液。称重10克，将其溶于100毫升MAB中，在微波炉中摇动加热1分钟(？) 葡萄膜保存25分钟，然后将其放入14毫升，然后保存在摄氏-20度。

 

绵羊血清(Sigma：S 2263)，ALI和储存于-20 oC

 

immnohybridization

 

加入1：2500(1：1000~1：5000)的新鲜封闭液，稀释碱性底物酶结合的地高辛抗体(抗地高辛-AP抗体片段)，在4 oC(12~20小时)孵育一夜。 。每管添加350~450μl，含0.03~0.04ml胚胎(0.1ml胚胎的1/3)。

 

抗地高辛-AP fab片段(来自绵羊)(1093274，BoehringerMannheim，现在的罗氏？)商店在4 OC，良好的超过1年半：自1999年1月19日)

 第五天

洗( wash的第三人称单数 )

 

室温下用MABT洗净，轻轻摇动。每隔45~60分钟更换一次溶液，室温下隔夜(12~18小时)。总之，必须至少改变8种解决方案。 时代.

 

抗体可保存在4℃，并可重复使用(尚未尝试)

 

第6天可视化

 

上一次用新鲜的MABT更换隔夜MABT洗涤液，开始准备平衡缓冲液。

 

用含左旋咪唑0.5mg/ml的平衡液(西格玛L-9756)改变mbt，孵育10分钟。同时，将胚胎移植到24孔组织培养板上(Falcon 3047，Mu)。 组织培养板)。

 

排出平衡缓冲液。加入含底物NBT/BCIP(4.5/3.5ul/ml)的新型平衡缓冲液(BoehringerMannheim，1681 451)和1.2mg/ml的左旋咪唑。用 将胚胎在室温下培养6小时(最多7小时)。如果缎纹持续12小时(最多48小时)，在感官控制实验中仍然没有背景，但是蛋黄贝克。 我不喜欢想象(当拍摄时，蛋黄看上去是黑色的，虽然你可以看到它在显微镜下观察时仍然是透明的)。

 

左旋咪唑(西格玛L-9756)：制成0.2mg/ul溶液，重量20毫克，溶解在100μl纳米水中。存放于4℃，并在7天内使用。

 

NBT/BCIP股票溶液(1681 451，Boehringer Mannheim，现在是Roche？)：在平衡缓冲液的5ml中加入100 ul的储存液。或者，

 

在70%二甲基甲酰胺(Mallinckrodt 4929)和50 mg/ml BCIP(760 994，Boehringer Mannheim，现在罗氏？)中4.5ul为75 mg/mlNBT(1087 479，Boehringer Mannheim，现在为Roche？) 将甲酰胺加入到1ml平衡缓冲液中。

 

平衡缓冲液：1ml 1mNaCl，1ml 1m Tris-HCL PH9.5 1ml 0.5M MgCl 2 7ml H2O 0.1%吐温-20