Axio imager m1 2

To visualize BDA, free-floating sections were washed in 125 mm PB three times for 10 min each. Endogenous peroxidases were blocked in three washes with 3% hydrogen peroxide in PB for 10 min each, followed by three washes in PB for 10 min each. The sections were subsequently washed in TBS-TX (50 mm Tris, 150 mm NaCl, 0.5% Triton X-100, pH 8.0) three times for 10 min each, followed by a 90-min incubation in TBS-TX with Alexa-conjugated streptavidin (Alexa Fluor 546, S11225, Invitrogen) in a 1:200 solution in TBS-TX overnight at 4°C. Then the sections were rinsed three times for 5 min in Tris-HCl and mounted on glass slides, air-dried overnight, cleared in toluene, and coverslipped with Entellan (Merck Darmstadt, catalog #107961). In some cases, Nissl staining was necessary to aid the delineation of brain regions.
To obtain digital images of all experimental material, we used automated slide scanners equipped with the appropriate brightfield and fluorescent imaging systems (Zeiss Axio Imager M1/2 and Zeiss Mirax Midi). We used 20× objectives (NA 0.8) and all files were postprocessed with the use of Adobe photoshop and illustrator (CS6, Adobe Systems).

To quantify the number of apoptotic corpses in the meiotic late pachytene germ cells in response to DNA damage, the synchronized L4 worms were separated from the rest of the population and 20 h later at the young adult stage were either remained untreated or exposed to 90 Gy of IR inflicted by a cesium 137 irradiation source (Biobeam GM 8000, Eckert & Ziegler, Gamma-Service Medical GmbH)^{3 (link)}. 6 h post-IR exposure, worms were immobilized using 5 mM levamisole (AppliChem #A431005) and mounted on a 2% agarose pad on a microscope slide. The number of apoptotic corpses was scored via Nomarski DIC microscopy on a Zeiss Axio Imager M1/2 based on the refractive morphological changes occurring in apoptotic germ cells within the gonad loop^{29 (link),63 (link)}. For quantification of heat stress-induced apoptosis, young adult worms on NGM plates were exposed for 10 min to heat shock at 35 °C in a pre-heated water bath and were treated with 30 Gy of IR. The number of apoptotic corpses was subsequently counted 5 h post-IR treatment via the same procedure as mentioned above.