Axiovision image capture software

Fixed cells were washed in several changes of phosphate buffered saline (PBS) before permeablisation and blocking in PBS containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100 (both from Sigma, Poole, UK). Cells were then incubated with monoclonal anti-βIII-tubulin antibody (Cat No. T8660; 1:200 dilution; Sigma) for 1 h at room temperature (RT) and used to localise RGC and their neurites. Cells were then washed in several changes of PBS and incubated with Alexa 488 anti-mouse IgG (Cat No. A-11001; 1:400 dilution; Invitrogen) for 1 h at RT, washed in several changes of PBS and mounted using Vectashield containing DAPI (Vector Laboratories, Peterborough, UK). Slides were then anonymised by a second investigator and viewed using a Zeiss epi-fluorescent microscope equipped with an AxioCam HRc and Axiovision Image capture software (all from Zeiss, Hertfordshire, UK). Immunocytochemistry included controls with primary antibody omitted, that were used to set background levels of nonspecific staining (not shown) prior to image capture.

Images were acquired using a Zeiss Axioplan microscope and Axiovision image capture software (v. 4.1). For each fish, one section at approximately the same point within the body was selected. In this randomly chosen section, supracarinalis area was measured by tracing the outside of this muscle group. Given the pronounced size differences between males and females in this species, muscle area was standardized to the standard length of each individual (muscle area (μm²) / standard length (mm)). In addition, two independent observers who were blind to the sex of the animal counted all AR positive supracarinalis muscle cells in each section. The number of AR cells was also counted within both the dorsal and ventral horns of the spinal cord. Additionally, the number of AR cells in swimming muscle was used as a control, as these muscles do not contribute to rapid starting and stopping or dorsal fin flexion. To perform these counts in epaxial and hypaxial muscles, we randomly chose a 400x300μm area of musculature and counted all AR+ cells within this area. This 120,000 μm² area approximates the average area of the supracarinalis muscle (both males and females combined, 156,009.8 ± 17,533.98). All cell counts and measurements were performed using ImageJ (National Institute for Health) on the previously acquired digital images.