Imaging and Quantitation of Assembly Dynamics of the Centromeric Histone H3 Variant CENP-A in Drosophila melanogaster Spermatocytes by Immunofluorescence and Fluorescence In-Situ Hybridization (Immuno-FISH)

Abstract

This chapter describes a method used to assay the cell cycle dynamics of the centromeric histone H3 variant CENP-A in meiosis using Drosophila males as the experimental system. Specifically, we describe a method that combines Immunofluorescence (IF) and Fluorescence in-situ Hybridization (FISH) protocols, performed on fixed Drosophila testes. An advantage of this protocol is the ability to localize individual centromeres on the four Drosophila homologous chromosomes that form distinct nuclear territories in spermatocytes. We also describe a method to quantify CENP-A focal intensities using Image J software.

Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy

“…Furthermore, an advantage of this combined protocol is that it can simultaneously detect various RNAs, proteins, and epigenetic modifications ( Ogawa and Ogawa, 2021 ). These combined protocols have also been applied to cultured mammalian cells ( Arrigucci et al, 2017 ; Eliscovich et al, 2017 ), and in other biological systems, such as Caenorhabditis elegans , zebrafish, and Drosophila, either in sections or whole-mount ( Toledano et al, 2012 ; Yoon et al, 2016 ; Collins and Dunleavy, 2018 ; He et al, 2020 ). Recently, such an approach has been used to detect retrotransposons in preimplantation mouse embryos using an smRNA FISH protocol with Stellaris probes ( Percharde et al, 2021 ).…”

Circular DNA vcDNA-S7 derived from segmented-dsRNA virus BmCPV attenuated viral infection through RNase H1

“…Finally, cell images were captured using a Leica DM2000 microscope (Leica, Germany). The ratio of red fluorescent cells was determined using the software Image J [26].…”