Axio observer a1 inverted

Epifluorescence animal imaging was performed on a ZEISS Axio Observer A1 inverted microscope affixed with objective lenses ranging from 10 to 100x magnification.
Confocal microscopy was performed in the Light Microscopy Core at the Max Planck Florida Institute for Neuroscience. Worms were mounted live on 2% agarose pad, anesthetized with 10% NaN₃ and imaged using an LSM Zeiss 780 confocal microscope. The Z-stack images were acquired at 1 μm slice intervals at 63X. GFP and mScarlet excitation/emission were set to 488/526 and 651/632 respectively and each laser was in an independent track. L1 stage and young adult were imaged 20 hr and 72 hr after egg-layer respectively. Dauers were generated as previously described and imaged after SDS selection.

Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were blocked with PBS containing 3% BSA and 0.2% Triton X-100 for 1 h. The cells were incubated with primary antibody (1:200, anti-SSRP1 or anti-CRE recombinase) in blocking solution for 1 h. The samples were washed three times in wash solution (6x dilution of blocking solution in PBS) for 10 min each and then incubated with secondary anti-mouse Alexa Fluor^® antibody (1:500) in washing solution for 45 min. The cells were washed three times in washing solution for 10 min each and then counterstained with Hoechst 33342 (1 μg/mL) for 10 min. Images were acquired using a Zeiss Axio Observer A1 inverted microscope with N-Achroplan 100x/1.25 oil lens, Zeiss MRC5 camera, and AxioVision Rel.4.8 software. Image analysis and quantification were performed using ImageJ software. Each image was divided into four quadrants, and ten cells in each quadrant were analyzed to determine the corrected total cell fluorescence (CTCF) using the following equation: CTCF = integrated density–(area of the selected cell x the mean fluorescence of background readings).