An Improved Staining Method for Low Signal LacZ Reporter Detection in Mouse Embryos
一种改进的可用于检测小鼠胚胎弱LacZ报告信号的染色法   

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PLOS ONE
May 2017

 

Abstract

In many fields of biology, especially in the field of developmental biology, LacZ reporter staining is an approach used to monitor gene expression patterns. In the LacZ reporter system, the LacZ gene is inserted in the endogenous location of the target gene via gene knock-in or by constructing a transgenic cassette in which LacZ is placed downstream of the promoter of the target gene being examined. Currently, the most common LacZ staining methods used are X-gal/FeCN staining and S-gal/TNBT staining. A serious limitation of both of these methods is that they are not effective when the LacZ gene is expressed at a low level. In an attempt to remedy this problem, we have established a new staining protocol which combines both methods. When compared to them, the method described here is better for visualizing lowly expressed genes and it has low background with high sensitivity.

Keywords: LacZ reporter (LacZ报告子), Mouse embryo (小鼠胚胎), Low expression (低表达), Low background (低背景), High sensitivity (高灵敏度)

Background

The LacZ gene has been widely used as a reporter gene for detecting gene expression patterns (Stevens et al., 1989; Bonnerot and Nicolas, 1993). The protein encoded by the LacZ gene is β-galactosidase, which is able to produce visible color when incubated with specific substrates. Usually, LacZ is placed downstream of a target gene promoter in lieu of the coding sequence of the gene. Therefore, visualizing LacZ expression patterns models the endogenous expression pattern of the target gene (Figure 1A). The most popular LacZ staining approach is the X-gal/FeCN method, in which β-galactosidase catalyzes X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) hydrolysis into 5-bromo-4-chloro-3-hydroxyindole and galactose. Then, 5-bromo-4-chloro-3-hydroxyindole is oxidized into an insoluble blue dimer with the help of potassium ferricyanide and potassium ferrocyanide, which finally displays blue color (Figure 1B) (Pearson et al., 1963; Lojda, 1970). While the X-gal/FeCN staining method is highly specific and has low background, it is unable to detect genes expressed at low levels due to the low sensitivity of this stain (Gugliotta et al., 1992; Sundararajan et al., 2012). The S-gal/TNBT staining method was developed to study genes with low expression. S-gal (6-Chloro-3-indolyl-β-D-galactopyranoside), a chromogenic substrate like X-gal, can be hydrolyzed by β-galactosidase and TNBT (Tetranitro Blue Tetrazolium) to produce dark-brown formazan compounds under reducing conditions (Figure 1C). The S-gal/TNBT method is more sensitive than the X-gal/FeCN one, but a drawback is that it can have strong non-specific background staining (Sundararajan et al., 2012).

By combining both the methods described above, we developed an improved LacZ reporter staining protocol that is highly sensitive and highly specific. This method adds an additional staining step from the S-gal/TNBT method to the X-gal/FeCN method. In our staining technique, the first staining step, which is from the X-gal/FeCN method, creates an oxidative environment which consumes non-specific enzymatic activity; the second step, which is from the S-gal/TNBT method, is a reaction that is specific for β-galactosidase and results in a sensitive and specific signal. The improved method described here has been validated by detecting several genes in different embryo stages. Furthermore, this method has been used to study relatively highly expressed genes with good results: strong and specific staining, with slightly higher background than the X-gal/FeCN method (Shen et al., 2017).


Figure 1. Schematic diagram of LacZ reporters and -Galactosidase reactions. (A) The LacZ reporter gene is placed downstream of the target gene's promoter. Thus, when the expression of the target gene is induced, the gene product of LacZ, β-galactosidase, is produced. The key steps of the enzymatic reactions in the X-gal/FeCN (B) and S-gal/TNBT (C) staining methods.

Materials and Reagents

  1. Sterile DNase/RNase free pipette tips 10 μl, 200 μl, 1,000 μl (Labselect, catalog numbers: T-001-10, T-001-200, T-001-1000)
  2. 6-well plates (NUNC, catalog number: 140675)
  3. Embryos collected from pregnant mice at E8.5, E9.5, and E10.5 stages. The mouse strain used here has been described previously in (Shen et al., 2017)
  4. Paraformaldehyde (PFA) (Sigma, catalog number: P6148)
  5. Nonidet P-40 (NP-40) (Sangon Biotech, catalog number: A510110)
  6. Sodium deoxycholate (Sigma, catalog number: D6750)
  7. X-gal (Gold Bio, catalog number: X4281C)
  8. K3Fe(CN)6 (Sigma, catalog number: 244023)
  9. K4Fe(CN)6 (Sigma, catalog number: P3289)
  10. MgCl2 (Sigma, catalog number: M8266)
  11. EGTA (Sigma, catalog number: E3889)
  12. IGEPAL (Sigma, catalog number: I8896)
  13. Salmon-gal (S-gal) (Lab Scientific, catalog number: X668)
  14. Tetranitro Blue Tetrazolium (TNBT) (VWR, catalog number: TCT0250)
  15. NaCl (Sigma, catalog number: S7653)
  16. KCl (Sigma, catalog number: P9333)
  17. Na2HPO4 (Sigma, catalog number: S7907)
  18. KH2PO4 (Sigma, catalog number: P5655)
  19. NaH2PO4 (Sigma, catalog number: S9638)
  20. PBS (see Recipes)
  21. 4% PFA (see Recipes)
  22. 0.1 M phosphate buffer (pH 7.3) (see Recipes)
  23. Wash buffer (see Recipes)
  24. Staining buffer 1 (see Recipes)
  25. Staining buffer 2 (see Recipes)

Equipment

  1. Pipettes (Gilson, catalog numbers: F144801, F123600, F144058M, F123602)
  2. Humidified chambers (for example, covered ice boxes or foam boxes with wet paper tower)
  3. Dissecting forceps (Fine Science Tools, model: Dumont #5)
  4. Stereomicroscope (Leica, model: MZ12.5)
  5. Orbital Shaker (MIULAB, GS-20)
  6. Microbiological Incubator (Thermo Scientific, PR305225G)
  7. Microscope (Leica, DFC340 FX digital FireWire Camera System)

Software

  1. Leica Application Suite V3.7 (Image taking software for Leica DFC340 FX digital FireWire Camera System)

Procedure

  1. Collect embryos from pregnant mice as reported previously (Shea and Geijsen, 2007) and fix the embryos in 10 ml 4% PFA (Recipe 2) in 6-well plates at room temperature (the volumes used here and in the following steps are for one embryo). The 6-well plates should be placed in humidified chambers, which are kept on orbital shakers set to 65 rpm. The fixation time varies and depends on the developmental stage of the embryos. The following table lists the recommended fixation times.


  2. Use forceps to transfer the embryos to a new well of the 6-well plate filled with 10 ml Wash Buffer (Recipe 4). Wash the embryos three times with a 10 min incubation at room temperature for each wash. During the washing steps, the 6-well plates should be placed in humidified chambers on an orbital shaker set at 65 rpm.
  3. Use forceps to transfer the embryos to a new well of the 6-well plate filled with 10 ml Staining Buffer 1 (Recipe 5). Incubate overnight at 37 °C in a microbiological incubator (remember to protect samples from light). The 6-well plates should be placed in humidified chambers.
  4. Use forceps to transfer the embryos to a new well of the 6-well plate filled with 10 ml Wash Buffer and incubate for 10 min at room temperature. Next use forceps to transfer the embryos to a new well of the 6-well plate with Staining Buffer 2 (Recipe 6) and incubate at 37 °C in a microbiological incubator. The 6-well plates should be placed in humidified chambers. The staining should be closely monitored with a stereomicroscope until specific staining appears. The staining time could vary from several minutes to one hour. 
  5. Use forceps to transfer the embryos to a new well of the 6-well plate filled with 10 ml Wash Buffer and wash three times. Incubate each wash for 10 min. During the wash, the 6-well plates should be placed in humidified chambers, on an orbital shaker set to 65 rpm. After the embryos are washed, they can be imaged directly or kept at 4 °C in Wash Buffer for up to one week. 
  6. Take images using Leica Application Suite V3.7 on a Leica DFC340 FX microscope. We use the Z-stack option in the Leica Application Suite V3.7 because the stained embryos are relatively thick. The images generated from the Z-stack processing are used as final results. Embryos that do not express the LacZ gene are used as negative controls. Figure 2 shows representative staining images of X-gal/FeCN, S-gal/TNBT, and the improved method described here.


    Figure 2. The representative LacZ staining images of X-gal/FeCN, S-gal/TNBT, and the improved method in detecting miR-322/-503’s expression in E10.5 embryos. A. Embryos stained with X-gal/FeCN method. B. Embryos stained with S-gal/TNBT method. C. Embryos stained with the improved method described here. The images were taken with a Leica DFC340 FX microscope at 10x magnification.

Data analysis

Scale bars, annotations, and arrows that point at the specific stained regions are added to the embryo images.

Recipes

  1. PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    in distilled water, pH = 7.4
  2. 4% PFA
    Dissolve 4% PFA in PBS
    Heat at 50 °C and vortex until fully dissolved
    Then adjust the pH to 7.2~7.4
  3. 0.1 M phosphate buffer (pH 7.3)
    23% NaH2PO4 solution (0.2 M NaH2PO4 in distilled water)
    77% Na2HPO4 solution (0.2 M Na2HPO4 in distilled water)
  4. Wash buffer
    0.02% NP-40
    0.01% sodium deoxycholate in PBS
  5. Staining buffer 1
    5 mM K3Fe(CN)6
    5 mM K4Fe(CN)6
    0.02% NP-40
    0.01% deoxycholate
    2 mM MgCl2
    5 mM EGTA
    1 mg/ml X-gal in PBS
  6. Staining buffer 2
    1 mg/ml S-gal
    0.4 mM TNBT
    0.1% sodium deoxycholate
    0.2% IGEPAL
    2 mM MgCl2 in 0.1 M phosphate buffer (pH 7.3)

Acknowledgments

We thank Wei Yu at the University of Houston for guidance in animal operations; Wenjing Bao at the Liaoning University of Traditional Chinese Medicine for technical support. This work was supported by the National Natural Science Foundation of China (No. 31701289), Anhui Provincial Natural Science Foundation (No. 1808085QH234), Educational Commission of Anhui Province of China (No. KJ2017A319), Foundation for High-level Talents in Higher Education of Anhui Province of China and Start-up Funds from the Anhui Normal University.

Competing interests

All animal-related work has been approved by the Institutional Animal Care and Use Committee (IACUC).

References

  1. Bonnerot, C. and Nicolas, J. F. (1993). Application of LacZ gene fusions to postimplantation development. Methods Enzymol 225: 451-469.
  2. Gugliotta, P., Pacchioni, D. and Bussolati, G. (1992). Staining reaction for β-galactosidase in immunocytochemistry and in situ hybridization. Eur J Histochem 36(2): 143-148.
  3. Lojda, Z. (1970). Indigogenic methods for glycosidases. I. An improved method for β-D-glucosidase and its application to localization studies on intestinal and renal enzymes. Histochemie 22(4): 347-361.
  4. Pearson, B., Wolf, P. L. and Vazquez, J. (1963). A comparative study of a series of new indolyl compounds to localize β-galactosidase in tissues. Lab Invest 12: 1249-1259.
  5. Shea, K. and Geijsen, N. (2007). Dissection of 6.5 dpc mouse embryos. J Vis Exp (2): 160.
  6. Shen, X., Bao, W., Yu, W., Liang, R., Nguyen, B. and Liu, Y. (2017). An improved method with high sensitivity and low background in detecting low β-galactosidase expression in mouse embryos. PLoS One 12(5): e0176915.
  7. Stevens, M. E., Meneses, J. J. and Pedersen, R. A. (1989). Expression of a mouse metallothionein-Escherichia coli β-galactosidase fusion gene (MT-β gal) in early mouse embryos. Exp Cell Res 183(2): 319-325.
  8. Sundararajan, S., Wakamiya, M., Behringer, R. R. and Rivera-Perez, J. A. (2012). A fast and sensitive alternative for β-galactosidase detection in mouse embryos. Development 139(23): 4484-4490.

简介

在许多生物学领域,特别是在发育生物学领域, LacZ 报告基因染色是一种用于监测基因表达模式的方法。 在 LacZ 报告系统中, LacZ 基因通过基因敲入或通过构建其中 LacZ的转基因盒插入靶基因的内源位置。 位于被检查的靶基因的启动子的下游。 目前,最常用的 LacZ 染色方法是X-gal / FeCN染色和S-gal / TNBT染色。 这两种方法的严重限制是当 LacZ 基因以低水平表达时它们无效。 为了解决这个问题,我们建立了一种结合两种方法的新染色方案。 与它们相比,这里描述的方法更好地可视化低表达的基因,并且它具有低背景和高灵敏度。
【背景】 LacZ 基因已广泛用作检测基因表达模式的报告基因(Stevens et al。,1989; Bonnerot和Nicolas,1993)。由 LacZ 基因编码的蛋白质是β-半乳糖苷酶,当与特定底物一起孵育时,其能够产生可见的颜色。通常, LacZ 位于靶基因启动子的下游,代替基因的编码序列。因此,可视化 LacZ 表达模式模拟靶基因的内源表达模式(图1A)。最流行的 LacZ 染色方法是X-gal / FeCN方法,其中β-半乳糖苷酶催化X-gal(5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷)水解成5-溴-4-氯-3-羟基吲哚和半乳糖。然后,在铁氰化钾和亚铁氰化钾的帮助下,将5-溴-4-氯-3-羟基吲哚氧化成不溶的蓝色二聚体,最终呈现蓝色(图1B)(Pearson et al。,1963; Lojda,1970)。虽然X-gal / FeCN染色方法具有高度特异性且背景低,但由于该染色剂的低灵敏度,它无法检测低水平表达的基因(Gugliotta et al。,1992; Sundararajan et al。,2012)。开发S-gal / TNBT染色方法以研究低表达的基因。 S-gal(6-氯-3-吲哚基-β-D-吡喃半乳糖苷),一种显色底物,如X-gal,可被β-半乳糖苷酶和TNBT(Tetranitro Blue Tetrazolium)水解,生成深棕色甲compound化合物。条件(图1C)。 S-gal / TNBT方法比X-gal / FeCN方法更敏感,但缺点是它可以具有强烈的非特异性背景染色(Sundararajan et al。,2012)。

通过结合上述两种方法,我们开发了一种改进的 LacZ 报告染色方案,该方案具有高度敏感性和高度特异性。该方法增加了从S-gal / TNBT方法到X-gal / FeCN方法的额外染色步骤。在我们的染色技术中,来自X-gal / FeCN方法的第一染色步骤产生消耗非特异性酶活性的氧化环境;第二步,来自S-gal / TNBT方法,是对β-半乳糖苷酶特异的反应,并产生敏感和特异的信号。这里描述的改进方法已通过检测不同胚胎阶段的几个基因得到验证。此外,该方法已用于研究相对高表达的基因,结果良好:强烈和特异性染色,背景略高于X-gal / FeCN方法(Shen et al。,2017)。


图1. LacZ 报告基因和β-半乳糖苷酶反应的示意图。 (A) LacZ 报告基因位于靶基因启动子的下游。因此,当诱导靶基因的表达时,产生 LacZ ,β-半乳糖苷酶的基因产物。 X-gal / FeCN(B)和S-gal / TNBT(C)染色方法中酶促反应的关键步骤。

关键字LacZ报告子, 小鼠胚胎, 低表达, 低背景, 高灵敏度

材料和试剂

  1. 无菌DNase / RNase移液器吸头10μl,200μl,1,000μl(Labselect,目录号:T-001-10,T-001-200,T-001-1000)
  2. 从E8.5,E9.5和E10.5阶段的怀孕小鼠收集的胚胎。此处使用的小鼠品系已在先前描述过(Shen et al。,2017)
  3. 多聚甲醛(PFA)(Sigma,目录号:P6148)
  4. Nonidet P-40(NP-40)(Sangon Biotech,目录号:A510110)
  5. 脱氧胆酸钠(西格玛,目录号:D6750)
  6. X-gal(Gold Bio,目录号:X4281C)
  7. K 3 Fe(CN) 6 (Sigma,目录号:244023)
  8. K 4 Fe(CN) 6 (Sigma,目录号:P3289)
  9. MgCl 2 (Sigma,目录号:M8266)
  10. EGTA(Sigma,目录号:E3889)
  11. IGEPAL(Sigma,目录号:I8896)
  12. Salmon-gal(S-gal)(Lab Scientific,目录号:X668)
  13. Tetranitro Blue Tetrazolium(TNBT)(VWR,目录号:TCT0250)
  14. NaCl(Sigma,目录号:S7653)
  15. KCl(Sigma,目录号:P9333)
  16. Na 2 HPO 4 (Sigma,目录号:S7907)
  17. KH 2 PO 4 (Sigma,目录号:P5655)
  18. NaH 2 PO 4 (Sigma,目录号:S9638)
  19. PBS(见食谱)
  20. 4%PFA(见食谱)
  21. 0.1 M磷酸盐缓冲液(pH 7.3)(见食谱)
  22. 洗涤缓冲液(见食谱)
  23. 染色缓冲液1(见食谱)
  24. 染色缓冲液2(见食谱)

设备

  1. 移液器(Gilson,目录号:F144801,F123600,F144058M,F123602)
  2. 6孔板(NUNC,目录号:140675)
  3. 加湿室(例如带盖的冰盒或带湿纸塔的泡沫箱)
  4. 解剖钳(精细科学工具,型号:Dumont#5)
  5. 立体显微镜(徕卡,型号:MZ12.5)
  6. 轨道振动筛(MIULAB,GS-20)
  7. 微生物培养箱(Thermo Scientific,PR305225G)
  8. 显微镜(Leica,DFC340 FX数字火线相机系统)

软件

  1. Leica Application Suite V3.7(Leica DFC340 FX数字FireWire相机系统的图像获取软件)

程序

  1. 如先前报道(Shea和Geijsen,2007)从怀孕小鼠收集胚胎,并在室温下将胚胎在10孔4%PFA(配方2)中固定在6孔板中(此处和以下步骤中使用的体积为一个胚胎)。 6孔板应放置在加湿室中,保持在设定为65rpm的轨道振荡器上。固定时间不同,取决于胚胎的发育阶段。下表列出了建议的固定时间。


  2. 使用镊子将胚胎转移到装有10ml洗涤缓冲液的6孔板的新孔中(配方4)。每次洗涤,在室温下孵育胚胎10分钟洗涤三次。在洗涤步骤期间,6孔板应放置在设定为65rpm的轨道振荡器上的加湿室中。
  3. 使用镊子将胚胎转移到装有10ml染色缓冲液1(配方5)的6孔板的新孔中。在微生物培养箱中37°C孵育过夜(记住保护样品免受光照)。 6孔板应放置在加湿室中。
  4. 使用镊子将胚胎转移到装有10ml洗涤缓冲液的6孔板的新孔中,并在室温下孵育10分钟。接下来使用镊子将胚胎用染色缓冲液2(配方6)转移到6孔板的新孔中,并在37℃下在微生物培养箱中孵育。 6孔板应放置在加湿室中。应使用立体显微镜密切监测染色,直至出现特异性染色。染色时间可能从几分钟到一小时不等。 
  5. 使用镊子将胚胎转移到装有10ml洗涤缓冲液的6孔板的新孔中并洗涤三次。孵育每次洗涤10分钟。在洗涤过程中,6孔板应置于加湿室中,在设定为65rpm的轨道振荡器上。清洗胚胎后,可将其直接成像或在4°C的洗涤缓冲液中保存一周。 
  6. 在Leica DFC340 FX显微镜上使用Leica Application Suite V3.7拍摄图像。我们在Leica Application Suite V3.7中使用Z-stack选项,因为染色的胚胎相对较厚。从Z-堆叠处理生成的图像用作最终结果。不表达 LacZ 基因的胚胎用作阴性对照。图2显示了X-gal / FeCN,S-gal / TNBT的代表性染色图像,以及此处描述的改进方法。


    图2. X-gal / FeCN,S-gal / TNBT的代表性 LacZ 染色图像,以及检测E10.5胚胎中miR-322 / -503表达的改进方法。 A.用X-gal / FeCN法染色的胚胎。 B.用S-gal / TNBT方法染色的胚胎。 C.用本文所述的改进方法染色的胚胎。用Leica DFC340 FX显微镜以10x放大率拍摄图像。



数据分析

将指向特定染色区域的比例尺,注释和箭头添加到胚胎图像中。

食谱

  1. PBS
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH 2 PO 4
    在蒸馏水中,pH = 7.4
  2. 4%PFA
    将4%PFA溶解在PBS中
    在50°C加热并涡旋直至完全溶解 然后将pH调节至7.2~7.4
  3. 0.1 M磷酸盐缓冲液(pH 7.3)
    23%NaH 2 PO 4 溶液(0.2M NaH 2 PO 4 在蒸馏水中)
    77%Na 2 HPO 4 溶液(0.2M Na 2 HPO 4 在蒸馏水中)
  4. 洗涤缓冲液
    0.02%NP-40
    PBS中的0.01%脱氧胆酸钠
  5. 染色缓冲液1
    5mM K 3 Fe(CN) 6
    5mM K 4 Fe(CN) 6
    0.02%NP-40
    0.01%脱氧胆酸盐
    2 mM MgCl 2
    5 mM EGTA
    PBS中1mg / ml X-gal
  6. 染色缓冲液2
    1毫克/毫升S-gal
    0.4 mM TNBT
    0.1%脱氧胆酸钠
    0.2%IGEPAL
    在0.1M磷酸盐缓冲液(pH 7.3)中的2mM MgCl 2,

致谢

我们感谢休斯顿大学的魏宇在动物经营方面提供指导;文静宝在辽宁中医药大学获得技术支持。国家自然科学基金(No. 31701289),安徽省自然科学基金(No. 1808085QH234),安徽省教育委员会(No. KJ2017A319),高等学历人才高等基金资助项目安徽省教育与安徽师范大学创业基金。

利益争夺

所有与动物有关的工作均已获得机构动物护理和使用委员会(IACUC)的批准。

参考

  1. Bonnerot,C。和Nicolas,J.F。(1993)。 将 LacZ 基因融合应用于植入后发育。 Methods Enzymol 225:451-469。
  2. Gugliotta,P.,Pacchioni,D。和Bussolati,G。(1992)。 免疫细胞化学和原位杂交中β-半乳糖苷酶的染色反应。< / a> Eur J Histochem 36(2):143-148。
  3. Lojda,Z。(1970)。 糖苷酶的靛蓝方法。 I.改进的β-D-葡萄糖苷酶方法及其在肠和肾酶定位研究中的应用。 Histochemie 22(4):347-361。
  4. Pearson,B.,Wolf,P。L.和Vazquez,J。(1963)。 对一系列新的吲哚基化合物进行比较研究,以定位组织中的β-半乳糖苷酶。 Lab Invest 12:1249-1259。
  5. Shea,K。和Geijsen,N。(2007)。 解剖6.5 dpc小鼠胚胎。 J Vis Exp (2):160。
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引用:Shen, X., Xu, F., Wu, S., Li, M., Zhang, J., Liang, R. and Liu, Y. (2019). An Improved Staining Method for Low Signal LacZ Reporter Detection in Mouse Embryos. Bio-protocol 9(5): e3180. DOI: 10.21769/BioProtoc.3180.
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