Tissue Processing and Anatomical Delineation of Visual Areas

The complete protocol for tissue fixation and CTb immunohistochemistry is described in detail elsewhere (Khalil and Levitt, 2014; Khalil et al., 2018). Briefly, animals were transcardially perfused using saline solution followed by a 4% paraformaldehyde solution, then a 4% paraformaldehyde plus 10% sucrose solution. The brains were removed from the skull and the posterior portion was blocked, and placed in a postfix solution of 4% buffered paraformaldehyde plus 30% sucrose for 2–3 h. The brains were then placed into a 0.1 M phosphate buffer (PB) solution with 30% sucrose for 2 days until they were sunk. Frozen 40 μm thick semi-tangential sections were cut using a sliding microtome. The sections were separated into four numbered series. The first and the third series were processed to reveal the CTb label using a modified version of the CTb protocol described by Angelucci et al. (1996). Sections from the remaining series were processed for cytochrome oxidase (CO) (Wong-Riley, 1989), Nissl substance, or synaptic zinc following the protocol previously described (Khalil and Levitt, 2013, 2017). Sections stained for CO, Nissl substance, and synaptic zinc reveal areal and laminar boundaries as previously described (Khalil and Levitt, 2013, 2014, 2017; Khalil et al., 2018), and were compared with adjacent CTb stained sections to assign tracer injection site and retrogradely labeled cells to particular areas and layers.