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Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae

Nature Protocols volume 15, pages 3361–3379 (2020)Cite this article

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Abstract

RNA fluorescence in situ hybridization (FISH) and antibody staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs and proteins. Here we describe a combined FISH and IF protocol to simultaneously detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. In our approach, FISH is performed before IF to prevent mRNA degradation during the IF procedure. Instead of proteinase K digestion, Triton X-100 treatment and skin removal are used to permeate tissues and preserve antigen epitopes, making this protocol applicable to both whole-mount embryos and larvae. Off-target hybridization and FISH background are reduced by using PCR-amplified DNA templates and stringent buffers. This protocol simultaneously detects multiple mRNAs and proteins with high sensitivity, and enables detection at single-cell resolution. The protocol can be completed within 6 days, overcoming the shortage of reliable antibodies available for zebrafish and exploiting the advantages of zebrafish for studying organ development and regeneration.

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Fig. 1: Summary of steps involved in the combination of FISH and antibody staining and the approximate time needed.
Fig. 2: Double FISH in combination with antibody staining of endoderm in early embryonic stage.
Fig. 3: The steps for skin removal.
Fig. 4: FISH in combination with double-antibody staining of endoderm organs in larvae.
Fig. 5: FISH in combination with antibody staining of liver and pancreatic β cells at single-cell-resolution in larvae.
Fig. 6: FISH in combination with antibody staining of meningeal lymphatic and vascular system in larvae.
Fig. 7: FISH in combination with antibody staining in muscle cells of late embryos and larvae.

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Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Any additional data, if needed, will be provided upon request.

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Acknowledgements

We thank K. D. Poss for providing pCS2-ctgfa:GFP plasmid. This work was supported by the National Key Research and Development Program of China (2017YFA0106600), the National Natural Science Foundation of China (31730060, 31970784, 31801214, 91739304) and the 111 Program (B14037).

Author information

Author notes

  1. These authors contributed equally: Jianbo He, Dashuang Mo.

Authors and Affiliations

  1. Institute of Developmental Biology and Regenerative Medicine, Southwest University, Beibei, Chongqing, China

    Jianbo He, Dashuang Mo, Jingying Chen & Lingfei Luo

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  1. Jianbo He
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Contributions

L.L., J.H. and D.M. designed the experimental strategy, analysed data and wrote the manuscript. J.C. performed the experiments shown in Fig. 6 and Extended Data Fig. 6. D.M. and J.H. performed all the other experiments.

Corresponding author

Correspondence to Lingfei Luo.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Uwe Strähle and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

He, J. et al. Hepatology 70, 2092−2106 (2019): https://doi.org/10.1002/hep.30790

Chen, J. et al. Dev. Cell 49, 697−710 (2019): https://doi.org/10.1016/j.devcel.2019.03.022

He, J., Lu, H., Zou, Q. & Luo, L. Gastroenterology 146, 789−800 (2014): https://doi.org/10.1053/j.gastro.2013.11.045

Extended data

Extended Data Fig. 1 FISH in combination with antibody staining of brain neuron and spinal cord at late embryonic and larval stage.

a, Labelling of FISH-huc (a gene specific to brain neurons and spinal cord), anti-GFP and DAPI in the whole body in the Tg(RasGFP) transgenic line at 4 dpf (3D stack of images; 448 µm, 32 slices, 14 µm each slice). b, Labelling of FISH-huc, anti-GFP and DAPI in the whole body in the Tg(RasGFP) transgenic line at 10 dpf (3D stack of images; 448 µm, 32 slices, 14 µm each slice). Note that huc mRNA is expressed in the brain neuron, spinal cord and intestine; the GFP and DAPI stained the whole body. Scale bar, 100 µm.

Extended Data Fig. 2 FISH in combination with antibody staining of notochord in larvae.

a, The Kaede expresses in the notochord at 5 and 10 dpf in the transgenic line Tg(ctgfa:Kaede). b, Labelling of FISH-ctgfa, anti-Kaede and DAPI in the notochord (dashed line) in Tg(ctgfa:Kaede) at 5 dpf (3D stack of images; 68 µm, 17 slices, 4 µm each slice). c, Labelling of FISH-ctgfa, anti-Kaede and DAPI in the notochord (dashed line) in Tg(ctgfa:Kaede) at 10 dpf (3D stack of images; 68 µm, 17 slices, 4 µm each slice). Scale bar, 100 µm.

Extended Data Fig. 3 FISH in combination with antibody staining of pectoral fin, skin and hair cells at 10 dpf.

a, The Tomato expresses in skin and hair cells at 10 dpf in the transgenic line Tg(krt18:Tomato). b, Labelling of FISH-ctgfa (a pectoral fin marker), anti-Tomato and DAPI in the pectoral fin (dashed line, top view) in Tg(krt18:Tomato) at 10 dpf (3D stack of images; 152 µm, 38 slices, 4 µm each slice; dorsal view). c, Labelling of FISH-ctgfa (a pectoral fin marker), anti-Tomato and DAPI in the pectoral fin (dashed line, front view) in Tg(krt18:Tomato) at 10 dpf (3D stack of images; 152 µm, 38 slices, 4 µm each slice; ventral view). Note that the Tomato expresses in the skin and FISH-ctgfa expresses in the pectoral fin under the skin. d, Labelling of FISH-krt18 (the skin and hair cells specific-gene), anti-Tomato and DAPI in skin and hair cells in Tg(krt18:Tomato) at 10 dpf (3D stack of images; 232 µm, 58 slices, 4 µm each slice). Note that the FISH-krt18 merges with Tomato and expresses strongly in hair cells (arrowheads) and weekly in the skin. BF, bright field. Scale bar, 100 µm.

Extended Data Fig. 4 FISH in combination with antibody staining of hepatic duct cells in the liver and secretory cells in the intestine at 20 dpf.

a–d, Labelling of FISH-anxa4 (the antigen of 2F11), 2F11 (the marker of hepatic duct cells and secretory cells in the intestine) and DAPI in hepatic duct cells in the liver and secretory cells in the intestine in wild type at 20 dpf (3D stack of images; 337.33 µm, 38 slices, 8.877 µm each slice; left view). BF, bright field. Scale bar, 100 µm.

Extended Data Fig. 5 Mounting the embryos or larvae.

a, A glass-bottom microwell dish (30 mm) was used for mounting the embryos and larvae. The samples were mounted in the middle of the dish with 100–120 µl 1.2% LMP agarose.

Extended Data Fig. 6 Comparison of the current protocol with the traditional FISH protocol combined with antibody staining in the liver and meningeal lymphatic and vascular system at the larval stage.

a, The liver after triple labelling of FISH-cp, 2F11, anti-GFP and with DAPI staining under the Tg(RasGFP) transgenic line at 5 dpf (3D stack of images; 104 µm, 26 slices, 4 µm each slice) using this protocol. b, The liver after triple labelling of FISH-cp, 2F11, anti-GFP and with DAPI staining under the Tg(RasGFP) transgenic line at 5 dpf (2D image) using this protocol. c, The liver after triple labelling of FISH-cp, 2F11, anti-GFP and with DAPI staining under the Tg(RasGFP) transgenic line at 5 dpf (3D stack of images; 108 µm, 27 slices, 4 µm each slice) using the traditional FISH protocol combined with antibody staining. d, The liver after triple labelling of FISH-cp, 2F11, anti-GFP and with DAPI staining under the Tg(RasGFP) transgenic line at 5 dpf (2D image) using the traditional FISH protocol combined with antibody staining. Note that the 2F11 and anti-GFP are hardly detectable with the traditional protocol. e, The meningeal lymphatic and vascular cells after triple labelling of the Dig-labelled mrc1a probe, anti-Kaede and anti-CFP in the Tg(lyve1b:Kaede; kdrl:CFP−NTR) transgenic line at 5 dpf using this protocol (3D stack of images; 200 µm, 50 slices, 4 µm each slice). f, The meningeal lymphatic and vascular cells after triple labelling of Dig-labelled mrc1a probe, anti-Kaede and anti-CFP in the Tg(lyve1b:Kaede; kdrl:CFP-NTR) transgenic line at 5 dpf using traditional FISH protocol combined with antibody staining (3D stack of images; 200 µm, 50 slices, 4 µm each slice). Note that the signals of FISH-mrc1a, anti-CFP and anti-Kaede are weak in the traditional protocol compared with this protocol. Scale bar, 100 µm.

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary References and Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Removing the skin of zebrafish embryo/larva. Use the tweezer in the left hand to hold the head of embryo/larva. Then, use the tweezer in the right hand to peel off the skin from the heart area. After removing the skin of the trunk from the heart to the tail fin, use tweezer in the left hand to hold the embryo/larva by the swim bladder. Then, dissect the skin from the hindbrain with the right hand, and remove all the skin from the head.

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He, J., Mo, D., Chen, J. et al. Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae. Nat Protoc 15, 3361–3379 (2020). https://doi.org/10.1038/s41596-020-0376-7

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