Protocol to study DNA strand breaks during development and apoptosis using in situ nick translation in Drosophila

Abstract

Cellular stress causes DNA strand breaks that are typically repaired to maintain homeostasis and regulate cell fate. However, unrepaired DNA breaks can be lethal, leading to cell death. Here, we present a protocol to study DNA strand breaks in Drosophila during development and apoptosis using in situ nick translation. We describe the steps for labeling DNA strand breaks using digoxigenin (DIG)-labeled nucleotide (DIG-11-dUTP) and visualizing them with anti-DIG immunostaining. We then detail procedures for mounting, imaging, and analysis. For complete details on the use and execution of this protocol, please refer to Maurya et al.¹ and Rigby et al.².

Keywords: Cell Biology; Developmental biology; Microscopy; Model Organisms.

Graphical abstract

Figure 1

Schematic representation of the step-by-step method details (A) Dissection of desired larval tissue in 1× PBS and removal of the debris. (B) Tissues were fixed in 4% PFA, immunostaining performed, and washed with 0.3% PBST thrice. Tissues were incubated in the nick-translation reaction mixture for 2 h at 37°C and washed thrice with 0.3% PBST. After incubation for 2 h in blocking solution, tissues were incubated in rhodamine-conjugated anti-DIG antibody solution containing DAPI for 2 h at 25°C. (C) After washing thrice with PBST, the lymph glands or eye discs were separated from the rest of the tissues, then mounted on the slide and observed under the confocal microscope.

Figure 2

In situ nick translation also labels the dying cells (A–D′) Nick positive cells (red) co-localized with γH2Av positive cells (green) in control lymph gland (e33c-Gal4/+) with nuclear DAPI staining (blue) (A and A′), without DAPI (B and B′), only nick translation (C and C′) and only γH2Av staining (D and D′). The lymph gland was dissected from wandering third instar larvae, and all images shown are single optical sections. The arrow indicates the co-localization of γH2Av staining and nick translation, and the arrowhead shows only nick translation. The scale bar represents 25 μm for the complete lymph gland lobe and 10 μm for all cropped high magnification images.

Figure 3

Numbers of nick-positive nuclei increase in the eye disc during cell death (A–A‴) A few nick-positive nuclei (red) were found in the control (GMR-Gal4/+) (n=15) eye disc (A). Also, shown in the red channel only (A′), and in high magnification (A″ and A‴). (B–B‴) Nick-positive nuclei (red) increase significantly upon apoptotic induction (GMR-hid) (n=14) (B). Also, shown in the red channel only (B′), and in high magnification (B″ and B‴). (C) Neurodegeneration also causes increased nick-positive nuclei (red) (GMR>127Q) (n=16) (C). Also, shown in the red channel only (C′), and in high magnification (C″ and C‴). (D) Absence of DNA polymerase I in the reaction mixture does not label any dying nuclei (GMR-hid), and it serves as a negative control of the experiment (D). Also, shown in the red channel only (D′), and in high magnification (D″ and D‴). (E) Quantification of nick-positive nuclei per eye disc (A–C‴). All eye discs dissected out from wandering third instar larvae, and all images shown are single optical sections. Nick translation marked in red and nuclei stained with DAPI (blue). The yellow line demarcated the GMR-positive area of the eye disc, and the white dotted square marks the region of the disc shown in high magnification. The scale bar represents 50 μm for all the eye disc images and 5 μm for all cropped high magnification images. ∗∗∗∗P < 0.0001 Error bars, mean ± SD. All images represent 3 or more independent biological experiments, and ‘n’ represents the number of lymph gland lobes.