An Improved Staining Method for Low Signal LacZ Reporter Detection in Mouse Embryos

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.