Ccd camera

Individual animals were photographed after 3, 4, 5, and 6 days from egg hatching using a Leica MZ10F stereomicroscope connected to Jenoptik CCD camera. Length of worm body was determined by using the Delta Sistemi IAS software. At least 30 nematodes were imaged on at least three independent experiments.

For the determination of the infection incidence on development and growth, nematode larvae that were infected with each bacterium, starting from embryos hatching, were photographed at the indicated time points by using a Leica MZ10F stereomicroscope with a Jenoptik CCD camera. The length of worm body was measured by using the Delta Sistemi IAS software. An average of 30 nematodes was imaged on at least three independent experiments.

Embryos and individual animals were photographed after 3, 4, and 5 days from egg hatching using a Leica MZ10F stereomicroscope connected to Jenoptik CCD camera. Length of embryos or worm body was determined by using the Delta Sistemi IAS software and compared to OP50- or LGG-fed worms. At least 30 nematodes or embryos were imaged on at least three independent experiments.

The Spatially-resolved Laser Activated Cell Sorter (SLACS) instrument comprises optical modules and mechanical modules for high-throughput retrieval of samples from the tissue. Two motorized stages exist for handling the retrieved targets. An X-Y axis motorized stage (ACS Motion Control, Migdal, Israel) was built to control the spatial location of the target. The device can be controlled automatically by communicating with a computer. One stage is for loading sample slides, and the other stage is for loading tubes to receive isolated cells. A charge-coupled device (CCD) camera (Jenoptik, Jena, Germany) was installed to observe where the laser pulse will be applied through the Objective lenses. A neodymium-doped yttrium aluminum garnet (Nd:YAG) nanosecond laser was purchased from Continuum (Minilite™ Series ML II; Continuum, San Jose, CA). There is a slit located in the light path between the laser source and the objective lens to control the region to be isolated. The slit is controlled either manually or automatically to adjust the size of the laser pulse. Objective lenses with various magnifications were purchased from Mitutoyo. The long working distance allows more space between the lens and the sample for user convenience.

Synchronized N2 worms were incubated at 16°C on NGM plates seeded with OP50 and 2-HIBA at the concentrations of 5, 10 or 20 mM, allowing embryos to lay. For fertility analysis, three individual animals were transferred onto a fresh plate every day, and the total number of progenies was counted with a Zeiss Axiovert 25 microscope. The procedure was repeated until the mother worms stopped laying eggs, at around day 6. Each day the progeny production was recorded, and we reported the sum from day 1 to day 6, which was finally compared with the untreated control. The experiment was performed three times.
For body length measures, animals were photographed from 1 to 5 days from egg hatching using a Leica MZ10F stereomicroscope connected to a Jenoptik CCD camera. Length of worm body was determined by using the Delta Sistemi IAS software and compared to untreated worms. At least 30 nematodes were analysed for each data set and at least three independent experiments were performed.

For γH2AX and RAD51 immunofluorescence, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and blocked with 1% normal goat serum (for γH2AX) or 3% BSA (for RAD51) in PBS for 1 h. For TOP1cc staining, cells were fixed with 4% paraformaldehyde for 15 min at 4 °C, permeabilized with 0.25% Triton X-100 in PBS for 15 min at 4 °C and treated with 1% SDS in PBS for 5 min at room temperature. Cells were washed five times with wash buffer (0.1% Triton X-100, 0.1% BSA in PBS) and blocked with 10% milk in 10 mM Tris-HCl pH 7.4 and 150 mM NaCl. Primary antibodies used were mouse anti-phospho-Histone H2A.X (Ser139) (Millipore, 05-636, 1:500), rabbit anti-RAD51 (Calbiochem, PC130, 1:300), and mouse anti-TOP1cc antibody^{53 (link)} (1:100). Secondary antibodies used were goat anti-mouse IgG Alexa fluor 488 (Invitrogen, A11029, 1:2000) and goat anti-rabbit IgG Alexa fluor 488 (Invitrogen, A11034, 1:2000). Images were captured using a Zeiss Axio Scope.A1 fluorescent microscope equipped with a CCD camera (Jenoptik). Foci were scored using ImageJ on images captured with a 63× objective (for γH2AX) or counted manually (for RAD51 and TOP1cc). At least 100 cells were scored for focus formation by blinded observers.