Bx51 microscope

10 µl of saturated E. coliOP50 was spotted to an NGM plate and allowed to dry. Fifteen 2-d-old unc-64(lf) hermaphrodites were transferred to the OP50 and allowed to incubate for at least 1 hr. The OP50 + NGM + hermaphrodites were then placed on a microscope slide. The behavior of one male at a time was recorded using an Olympus BX51 microscope and Hamamatsu ImagEM Electron multiplier (EM) CCD camera. Males were given 10 min to insert their spicules into the hermaphrodite uterus, after which the mating trial was terminated. Spicule insertion % was determined by calculating the number of males that were successfully able to insert their spicules within 10 min vs. the total number of males tested. Sperm release from the valve % was determined using the total number of males that inserted. All males that were able to release some sperm from the valve were counted as successful whether or not they were able to release sperm from the cloaca. Sperm release measures all males that successfully transferred sperm into the uterus vs. the total number of males that inserted their spicules. The total amount of time that males’ spicules were inserted measures from when they inserted their spicules until they either retracted their spicules into their cloaca or removed the still-protracted spicules from the uterus. The competition assay was done as described in LeBoeuf et al. (2011) (link).

Images were taken with either an Olympus BX51 microscope and Hamamatsu ImagEM Electron multiplier (EM) CCD camera or with a Olympus IX81 microscope, csu-xi Yokogawa spinning disk, and Andor iXon EM CCD camera.

A small drop of blood was collected from the frog foot with a sterile needle, and the drop was smeared on a slide. The smear was then fixed with methanol and stained with Giemsa stain (Sigma GS). Cell were imaged in brightfield using micromanager software (Edelstein et al., 2014 (link)) with an Olympus BX51 microscope equipped with an ORCA-II camera (Hamamatsu Photonics, Hamamatsu city, Japan).

For immunofluorescence staining, cells were cultured in Millicell EZ 8-well chamber slides (Merck Millipore, Watford, UK) for 18 h at a seeding density of 5 × 10⁴ cells per well. Cells were fixed in 500 μl of ice cold 100% ethanol at −20 °C. Cells were permeabilised with 0.1% Triton X 100 (Sigma Aldrich). Primary antibodies (diluted to 1:100) and secondary antibodies (1:500 for Alexa secondary antibodies and 1:1000 for DAPI) were prepared in blocking buffer in blocking buffer and applied. Slides were washed and mounted using FluorSave (Calbiochem, Nottingham, UK) and visualised using an Olympus BX51 microscope with a Hamamatsu Orca ER digital camera at × 40. Integrated density was measured using ImageJ.