Enhancing protein signal detection in asexual and viviparous pea aphids: A guided protocol for tissue dissection and proteinase K treatment
Chun-wei Lai,
Gee-Way Lin,
Wen-Chih Lee,
Chun-che Chang
Affiliations
Chun-wei Lai
Laboratory for Genomics and Development, Department of Entomology, College of Bio-Resources and Agriculture, National Taiwan University (NTU), Taipei, Taiwan; Genome and Systems Biology Degree Program, NTU, Taipei, Taiwan; Research Center for Developmental Biology and Regenerative Medicine, NTU, Taipei, Taiwan; Taiwan Aphid Genomics Consortium, MK Innovation Hall, NTU, Taipei, Taiwan
Gee-Way Lin
Laboratory for Genomics and Development, Department of Entomology, College of Bio-Resources and Agriculture, National Taiwan University (NTU), Taipei, Taiwan; College of Medicine, Taipei Medical University, Taipei, Taiwan; Research Center for Developmental Biology and Regenerative Medicine, NTU, Taipei, Taiwan; Taiwan Aphid Genomics Consortium, MK Innovation Hall, NTU, Taipei, Taiwan
Wen-Chih Lee
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; Taiwan Aphid Genomics Consortium, MK Innovation Hall, NTU, Taipei, Taiwan
Chun-che Chang
Laboratory for Genomics and Development, Department of Entomology, College of Bio-Resources and Agriculture, National Taiwan University (NTU), Taipei, Taiwan; Genome and Systems Biology Degree Program, NTU, Taipei, Taiwan; Institute of Biotechnology, College of Bio-Resources and Agriculture, NTU, Taipei, Taiwan; Research Center for Developmental Biology and Regenerative Medicine, NTU, Taipei, Taiwan; International Graduate Program of Molecular Science and Technology, NTU, Taipei, Taiwan; Master Program for Plant Medicine, NTU, Taipei, Taiwan; Taiwan Aphid Genomics Consortium, MK Innovation Hall, NTU, Taipei, Taiwan; Corresponding author at: Laboratory for Genomics and Development, Department of Entomology, College of Bio-Resources and Agriculture, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106319, Taiwan.
Aphids, as hemipteran insects, reproduce via parthenogenesis and viviparity, resulting in rapid and exponential offspring production. To investigate the molecular mechanisms underlying parthenogenetic viviparity in asexual aphids, precise protein detection through immunostaining is essential. Our previous research demonstrated the need for proteinase K (PK) treatment to improve tissue permeability, enabling antibodies targeting the germ-cell marker Ap-Vas1 to access gastrulating and later-stage embryos. However, optimal PK digestion protocols have not been thoroughly explored. In this study, we propose strategies to optimize PK digestion conditions for early, middle, and late-stage pea aphid embryos, which have varying tissue thicknesses. Additionally, we extend the application of PK treatment to salivary glands, a representative somatic tissue, by optimizing conditions for antibody penetration against the salivary gland marker C002. To enhance spatial precision in signal detection, we provide a detailed protocol for tissue dissection specific to pea aphids, focusing on the preservation of tissue integrity. These comprehensive guidelines, covering tissue dissection and PK titration, are expected to improve the specificity and intensity of protein signals in pea aphids and other aphid species. • Provide aphid-specific dissection methods to obtain intact embryos and salivary glands. • Present strategies for optimizing PK treatment conditions across different tissue types.
