Axioplan fluorescent microscope

Synchronized worms at the relevant age were immobilized on slides with 1 mM levamisole and imaged on a Zeiss Axioplan fluorescent microscope. All conditions that are quantitatively compared were imaged on the same day using the same microscope and image capture settings. Representative images of conditions that are not quantitatively compared may have been taken using different settings in order to best display the expression pattern. The average pixel intensity in the uterus of each worm was quantified using ImageJ [64 ] and background subtracted using the average pixel intensity of at least two representative background areas outside the worm. Regions of interest were selected manually.

Degenerating cells were determined in 10 µm thick paraffin sections collected at the hippocampal level (−3.12–−3.36 mm from bregma) using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Staining was performed using the In Situ Cell Death Detection Kit (Roche applied science, Penzberg, Germany) according to manufacturer's protocol. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Invitrogen, Karlsruhe, Germany) and slides were mounted with Fluorescent Mounting Medium and kept in the dark at 4°C. Immunopositive cells were counted manually in three regions of interest in the parietal cortex and the entire hippocampus by an observer, unaware of the treatment protocol, with an Axioplan fluorescent microscope (Zeiss, Jena, Germany) at 20x (cortex) and 10x (hippocampus) magnification (Supplemental Figure 2).

For each experiment, 30 worms were grown on OP50-1 carrying plasmid pFPV25, which expresses GFP under the control of rpsM. At each time point, worms were transferred to plates without bacteria for one hour, and then placed on agarose pads on slides with Levamisole (25 mM) to paralyze the worms. GFP levels from intact bacteria in the intestine were measured by fluorescence microscopy on a 20× lens on a Zeiss AxioPlan Fluorescent Microscope. Quantification of the fluorescence in each worm was performed using ImageJ. The number of worms displaying GFP in their pharynx and intestinal track was also scored.

Following exposure to neomycin and perturbagen compound dose responses, zebrafish larvae (n = 9–11 larvae per treatment group, aged 5–7 dpf) were fixed with 4% PFA (in 1X PBS) for 2 h at RT, followed by three 15-min washes in 1X PBS. Zebrafish larvae were then incubated for a 2-h blocking period at room temperature (1% Triton X-100, 5% NGS in PBS). Larvae were then immunostained with mouse anti-parvalbumin primary antibody (monoclonal 1:400, Millipore MAB1572) in primary block (1% Triton X-100, 1% NGS in 1X PBS) at 4°C overnight. Following three 15-min washes in PBS-T (1X PBS, 1% Triton X-100), larvae were transferred into a solution containing a goat anti-mouse secondary antibody conjugated to Alexafluor-488 (1:500) in secondary block (1% NGS in 1X PBS). Larvae were washed in three 15-min washes with PBS-T followed by three 15-min washes in 1X PBS. Larvae were mounted using Fluoromount G on glass slides, and hair cell counts were performed on the SO1, SO2, O1, and OC1 neuromasts using an Axioplan fluorescent microscope (Zeiss) at 40× magnification as previously described (Raible and Kruse, 2000 (link); Harris et al., 2003 (link)).

Antibody staining was performed as previously described [47 (link)]. In brief, L1 worms were collected in 100 μl M9, fixed with 200 μl of cold 2X witches brew and 100 μl 10% paraformaldehyde, and then incubated at 4°C for 30 min to overnight. The worms were washed twice in Tris-Triton buffer, incubated in 1% ßME/Tris-Triton for 1-2 hours at 37 °C, and then washed in 1X Borate buffer. Subsequently, the worms were incubated in 10 mM DTT/1X Borate buffer for 15 min at room temperature, washed in 1X Borate buffer, incubated in 0.3% H₂O₂/1X Borate buffer for 15 min, incubated for 15 min in PBST-B, and then washed with PBST-A. The worms were then visualized using an Axioplan fluorescent microscope (Zeiss, Germany).
For dpMPK-1 observation and antibody staining, the L1-arrested worms were collected at 10, 20, 40, and 48 hours after the embryos were prepared. We found that 40 hours after the embryos were placed in M9 buffer was the best time to detect dpMPK-1. Worms with more than two cells detected using anti-dpMPK-1 were scored. The anti-dpMPK-1 [26 (link)] and all secondary antibodies were purchased from Sigma-Aldrich (USA).