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 T 10 ul per ml of hyb-

Heparin (Sigma H3393): dissolve 50 mg heparin in 1 ml nanopure water (50 mg/ml); store at -20oC T 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（翻譯）

 

第一天

胚胎準(zhǔn)備。在晚上將胚胎收集器放入水箱。

第二天

早上收集胚胎。胚胎是清洗和清潔　解決胚胎在預(yù)期的階段。

固定

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

　　10×PBS股票的解決方案:76克氯化鈉混合,9.9 g Na2HPO4,3.6 g NaH2PO4。溶解在不到1升nanopure水,適應(yīng)pH7.0和最終體積的1升。高壓蒸汽消毒35分鐘。

　　130毫米氯化鈉= 10×0.13 X 58.44 = 76克氯化鈉

　　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很難溶解。)孵化混合物在65 oc 2小時左右。孵化期間搖混合物)。溶解后,解決方案是儲存在4攝氏度。3天(72小時)內(nèi)使用它。

　　

　　廢水盡可能。固定劑直接添加到1.5毫升埃普多夫微型離心機管。用新鮮的固定劑替代解決方案。重復(fù)3或4次。最后,最終以100%的固定劑。

　　

一般來說,固定胚胎在一夜之間將在4攝氏度,允許固定過程完整模型

 

脫水胚胎根據(jù)年齡如下:

　　1細(xì)胞80%外包:

　　步驟1:25%甲醇/ 75%固定劑在室溫下5分鐘

　　步驟2:50%甲醇/ 50%固定劑在室溫下5分鐘

　　步驟3:100%甲醇在室溫下5分鐘

　　進(jìn)行步驟1,吸入1/4的固定劑,添加相同量甲醇進(jìn)入管;第二步,拿出一半的解決方案在管,添加相同量甲醇進(jìn)入管;步驟3,去除大部分的解決方案的管,添加新鮮甲醇,重復(fù)2次,然后最終以100%甲醇。

　　小心把胚胎,胚胎的早期階段(balstula)是非常脆弱的。

胚胎12小時及以上:

　　步驟1:50%甲醇/ 50%固定劑在室溫下5分鐘

　　步驟2:100%甲醇在室溫下5分鐘

　　

　　間隙

　　存儲胚胎在-20 oc使用前至少1小時。在這一步中,胚胎可能永遠(yuǎn)是安全的。(幾個月絕對是好的。)

第三天

再水化，再水合

 

按年齡清洗胚胎如下：

 

1個細(xì)胞至80%表觀：

 

第一步：25%PBST/75%甲醇，室溫下5分鐘。

 

步驟2：50%PBST/50%甲醇在室溫下作用5分鐘

 

步驟3：室溫下100%PBST 5分鐘，兩次

 

對于步驟1，吸出1/4的甲醇，將相同量的pbst加入到管道中；在步驟2中，取出一半的溶液，在管道中加入相同量的pbst；對于步驟3，reMO。 把大部分溶液從管子里拿出來，加入新鮮的PBST，再重復(fù)一次。

 

小心的處理胚胎，胚胎的早期階段(到壓載期)是非常脆弱的。

 

12小時及以上胚胎：

 

步驟1：50%PBST/50%甲醇在室溫下5分鐘

 

步驟2：100% PBST在室溫下5分鐘，兩次

 

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

 

蛋白酶K處理

 

取下洗滌后的PBST，加入新的新鮮PBST至1.5ml，將所需蛋白酶K直接加入到試管中，輕輕搖動試管，以增加蛋白酶K的擴散。

（渦流管并使S 在使用前，將沉淀物均勻混合。

 

按年齡分列如下：

 

1細(xì)胞至20小時：不治療。

 

>24小時：室溫下10微克/毫升，持續(xù)7-8分鐘。

 

>36小時：室溫下10微克/毫升，10-12分鐘。

 

蛋白酶K(Sigma P-2308)；原液(10 mg/ml=10μg/ul)：稱重10 mg蛋白酶K，溶解于1ml納米水中。每箱50升，以攝氏-20度儲存。在室溫下解凍 使用。長時間的儲存可能會在解凍后出現(xiàn)白色沉淀。旋轉(zhuǎn)管道，確保沉淀物均勻混合。

 

售后：消解

 

取出溶液，用PBST沖洗胚胎，用新鮮的PBST在室溫下洗5分鐘。

 

posterization多色調(diào)分色法

 

取出PBST，加入固定劑(4%PFA，室溫加熱)到管子上，取出PFA，再加入新鮮的PFA。室溫下將胚胎固定20分鐘。

 

室溫下用PBST洗兩次5分鐘。

 

雜交

 

使用1.5毫升離心管

 

去除pbst，在55 oC(50-65 oC)下，加入Hyb-到預(yù)雜交胚胎5分鐘。

 

取出Hyb-，加入Hyb，在55℃孵育4小時(最多6小時)。

 

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

 

用高壓釜35分鐘調(diào)節(jié)pH=7進(jìn)行消毒。

 

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

 

Hyb-，500μg/ml酵母RNA，50μg/ml肝素。

 

甲酰胺(Fisher BP228-100，超純，用混合樹脂-去離子處理)

 

酵母RNA(Sigma R 6750)：將50毫克RNA溶于1毫升納米水(50毫克/毫升)；儲存于-20 oC(每毫升海布10微升)。

 

肝素(Sigma H 3393)：將50毫克肝素溶解于1毫升納米水(50毫克/毫升)；儲存于-20 oC(每毫升海布1微升)。

 

將探針加入1 ng/L的新鮮HYB+中，在65℃下加熱10分鐘，在冰上冷卻5分鐘，然后除去預(yù)雜交HYB+，并加入含有探針的新鮮HYB+（GED標(biāo)記）。 胚胎/100 ul Hyb

第四天

 

探針去除

 

探針可以在有限的時間內(nèi)重復(fù)使用，當(dāng)存儲在-20攝氏度時。結(jié)果表明，如果儲存時間超過2個月，重復(fù)使用的探針本底很高，但在一周內(nèi)重復(fù)使用，效果很好。 .

 

洗滌：所有步驟在攝氏60度(攝氏60度至攝氏65度)

 

2次，60℃，2×SSCT，50%甲酰胺(0.5福爾馬胺，0.1 20×SSC，0.1%)每毫升吐溫1μl，每次30 min。

 

1次，15分鐘，2×SSCT

 

0.2XSSCT(2 XSSCT/H2O 1/9)2次30分鐘

 

室溫預(yù)塊洗滌

 

3次使用MABT 5分鐘

 

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）

 

加入固體(？g)調(diào)節(jié)pH=7.5，高壓釜35分鐘進(jìn)行殺菌。

 

布洛克，赫伯特·勞倫斯（（生于 1909） 美國卡通片制作者，優(yōu)秀作品曾見諸<華盛頓郵報> 及遍布全國的200多家報紙。他獲得了1942年和1954年的普利策獎）

 

用2%封閉液、10%羊血清和70%MAB阻斷1小時(至4h)。每1.5毫升管加約1毫升。

 

阻擋攝政者(1096 176年，博林格曼海姆，現(xiàn)在羅氏？)10%的庫存溶液。稱重10克，將其溶于100毫升MAB中，在微波爐中搖動加熱1分鐘(？) 葡萄膜保存25分鐘，然后將其放入14毫升，然后保存在攝氏-20度。

 

綿羊血清(Sigma：S 2263)，ALI和儲存于-20 oC

 

immnohybridization

 

加入1：2500(1：1000~1：5000)的新鮮封閉液，稀釋堿性底物酶結(jié)合的地高辛抗體(抗地高辛-AP抗體片段)，在4 oC(12~20小時)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(來自綿羊)(1093274，BoehringerMannheim，現(xiàn)在的羅氏？)商店在4 OC，良好的超過1年半：自1999年1月19日)

 第五天

洗( wash的第三人稱單數(shù) )

 

室溫下用MABT洗凈，輕輕搖動。每隔45~60分鐘更換一次溶液，室溫下隔夜(12~18小時)?？傊仨氈辽俑淖?span style="font-family:calibri;">8種解決方案。 時代.

 

抗體可保存在4℃，并可重復(fù)使用(尚未嘗試)

 

第6天可視化

 

上一次用新鮮的MABT更換隔夜MABT洗滌液，開始準(zhǔn)備平衡緩沖液。

 

用含左旋咪唑0.5mg/ml的平衡液(西格瑪L-9756)改變mbt，孵育10分鐘。同時，將胚胎移植到24孔組織培養(yǎng)板上(Falcon 3047，Mu)。 組織培養(yǎng)板)。

 

排出平衡緩沖液。加入含底物NBT/BCIP(4.5/3.5ul/ml)的新型平衡緩沖液(BoehringerMannheim，1681 451)和1.2mg/ml的左旋咪唑。用 將胚胎在室溫下培養(yǎng)6小時(最多7小時)。如果緞紋持續(xù)12小時(最多48小時)，在感官控制實驗中仍然沒有背景，但是蛋黃貝克。 我不喜歡想象(當(dāng)拍攝時，蛋黃看上去是黑色的，雖然你可以看到它在顯微鏡下觀察時仍然是透明的)。

 

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

 

NBT/BCIP股票溶液(1681 451，Boehringer Mannheim，現(xiàn)在是Roche？)：在平衡緩沖液的5ml中加入100 ul的儲存液。或者，

 

在70%二甲基甲酰胺(Mallinckrodt 4929)和50 mg/ml BCIP(760 994，Boehringer Mannheim，現(xiàn)在羅氏？)中4.5ul為75 mg/mlNBT(1087 479，Boehringer Mannheim，現(xiàn)在為Roche？) 將甲酰胺加入到1ml平衡緩沖液中。

 

平衡緩沖液：1ml 1mNaCl，1ml 1m Tris-HCL PH9.5 1ml 0.5M MgCl 2 7ml H2O 0.1%吐溫-20