Dmra microscope

The tissues were fixed in paraformaldehyde (4% in PBS), dehydrated in graded ethanol, cleared, and embedded in paraffin. Then, 10 µm thick sections were obtained and mounted on the slides. The slides were deparaffinized and stained with hematoxylin (Sigma, St. Louis, MO, USA; MHS16) and eosin (Sigma, St. Louis, MO, USA; 2853), as per the standard procedure. Sections were observed under a Leica DMRA microscope (Leica Microsystems, Wetzlar, Germany). For immunohistochemistry, the slides were deparaffinized and rehydrated with graded alcohol. After quenching the endogenous peroxidase, the tissue sections were blocked with blocking serum (VECTASTAIN Elite ABC Kit; Vector Laboratories, New York, NY, USA; PK-6102). Tissue sections were incubated with an anti-Ki-67 primary antibody (1:1000, Ki-67 mouse mAb; Cell Signaling, Danvers, MA, USA; 9449) overnight at 4 °C. The tissue sections were washed and detected using a secondary antibody from VECTASTAIN Elite ABC Kit (Vector Laboratories, New York, NY, USA; PK-6102). The images were captured with a Leica DMRA microscope (Leica Microsystems, Wetzlar, Germany) and the staining was quantified by ImageJ software (ImageJ version 1.45, NIH, Bethesda, MD, USA).

Inhibitor exposure was performed in liquid media as described in the Liquid-Based Kinase Inhibitor Screening method above. Analysis was performed once all F0 worms reached adulthood (Day 4). For the MEK inhibitor experiments, worms were mounted on a 2% agarose pad on a glass slide, observed using a 20 × objective on a Leica DMRA microscope and the number of vulval protrusions were counted. For the EGFR inhibitor experiment, the adult worms were examined under a dissection scope and the vulva phenotype was categorized as Multivulva, WT or Vulvaless. F0 worms that contained hatched progeny trapped inside (bag-of-worms phenotype) were assumed to not have a functioning vulva and were counted as Vulvaless. A minimum of three biological replicates were completed for all vulva phenotype analyses.