For fixation, isolated cortices were treated for 5 min in 2–3% formaldehyde in CIB; coelomocytes were fixed following the methods of Henson et al. (1999) (link). For immunolocalization of RhoA, cortices were fixed using 10% trichloroacetic acid after Yonemura et al. (2004) (link). For blocking buffer, phosphate-buffered saline plus 2% goat serum and 1% bovine serum albumin was used, followed by incubation in appropriate primary and secondary antibodies. Cells were mounted in nonhardening Vectashield antifade mounting medium (Vector Laboratories, Burlingame, CA), and conventional epifluorescence microscopic screening of samples was performed on a Nikon (Tokyo, Japan) 80i microscope using a 60×/1.4 numerical aperture (NA) Plan Apochromatic phase contrast objective lens with digital images captured using a Photometrics CoolSnap Cf cooled charge-coupled device (CCD) camera (Roper Scientific, Tucson, AZ). For superresolution 3D SIM (Kner et al., 2009 (link)), we used a DeltaVision OMX 3D-SIM Imaging System (GE Healthcare Bio-Sciences, Pittsburgh, PA) with an Olympus (Tokyo, Japan) 60×/1.42 NA objective lens. Captured images were reconstructed using SoftWoRx software and additional linear adjustments and manipulations made using ImageJ and Photoshop (Adobe, San Jose, CA).