Optimized Lens Fixation and Immunolabeling

Lenses were dissected and immediately fixed as previously reported.²⁵ (link) For α-SMA labeling, a protocol employed by Nowak and colleagues,²⁶ (link) was adapted, and optimized to fix the lenses without dissection from the eye. To do this, a small incision was first made at the corneal limbus and posterior pole of the enucleated eye, before placing the whole eye in 1% w/v PFA for 24 hours. Fixed lenses were dissected from the eye, cryoprotected, sectioned, and labeled as previously described.²⁵ (link) For α-SMA labeling, sections were washed, and then incubated with α-SMA antibodies (1:200; Sigma-Aldrich, Darmstadt, Germany) overnight at 4°C. Sections were then labeled with Alexa Fluor 488 (1:200; Life Technologies, Carlsbad, CA, USA), washed, and then incubated in 4',6-diamidino-2-phenylindole (DAPI; 1:1000) and wheat germ agglutinin conjugated to an Alexa Fluor 594 (WGA; 1:100). Sections were then mounted with VECTASHIELD HardSet aqueous mounting medium (Vector Laboratories, Burlingame, CA, USA), and imaged using the Olympus FV1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). Raw data were processed and merged if required using Adobe Photoshop CS6 version 13.0 (Adobe Systems Pty. Inc., San Francisco, CA, USA).