Twenty whole globes from 10 adult mice from two inbred strains (five C57BL/6 and five BALB/c) were enucleated and fixed in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) for 4 hours, washed in PBS, and embedded in paraffin. Sagittal sections (5 μm) were cut and stained as described below. To investigate regional differences, an additional 12 eyes from three C57BL/6 and three BALB/c mice were marked by placing a small suture through the superior or temporal quadrants. For each pair of eyes, one eye was cut along the nasal-temporal axis, while the contralateral eye was cut along the superior-inferior axis. For whole-mount preparations, eight additional eyes from two C57BL/6 and two BALB/c mice were bisected sagittally under a stereomicroscope, the lens carefully removed, and the iris lifted to visualize and cut the pectinate ligaments near their insertion at the cornea. The iris and ciliary processes were carefully separated from the CM and outer TM, and placed on a slide with the inner lamellated TM facing the microscope objective. The remaining specimen contained the JCT, outer lamellated TM and most of the CM, and was placed on a slide with the JCT facing the microscope objective. For depigmentation, whole mounts were bleached with 1% hydrogen peroxide for 4 to 6 hours.
The specimens were incubated in BLOTTO's Blocking Buffer (Thermo Scientific, Waltham, MA, USA) at room temperature for 1 hour to reduce nonspecific background staining. Following three washes in Tris-buffered PBS, specimens were incubated with the primary antibody (
Table) at 4°C overnight, washed three times with PBS, and incubated with a secondary antibody (Alexa 488 or 555) for 2 hours. For double immunolabeling, specimens were washed three times with PBS, incubated with the second primary antibody (
Table) at 4°C overnight, washed three times with PBS and incubated with secondary antibody (Alexa 555 or 488) for 2 hours. The slides were examined with a Keyence Biorevo BZ9000 microscope (Keyence, Neu-Isenburg, Germany).
Antibody against α-smooth muscle actin (αSMA) was used to visualize contractile cells, vesicular acetylcholine transporter (VAChT) to visualize cholinergic nerves, tyrosine hydroxylase (TH) to visualize sympathetic nerves, neuronal nitric oxide synthase (nNOS), and vasoactive intestinal peptide (VIP) to visualize parasympathetic fibers of the facial nerve. Antibody details are given in the
Table.