Pureblu

The method used for immunofluorescent (IF) staining has been described previously.²⁵ (link) Briefly, the LGs and eyeballs were harvested and cryopreserved by embedding in optimal cutting temperature compound before staining. The histologic sections (5–7 µM) were collected on poly-L-lysine-coated slides and blocked with rabbit, goat, or rat serum followed by primary antibody conditioning for 40 minutes at room temperature and exposed to the following primary antibodies: FITC-conjugated DR1 (rat monoclonal anti-mouse, 2 µg/mL; Abcam, Cambridge, UK), DR2 (rabbit polyclonal anti-mouse, 1 µg/mL; Abcam), and mouse tyrosine hydroxylase (TH) (rat monoclonal anti-mouse, 2 µg/mL; ThermoFisher Scientific, Inc., Waltham, MA, USA). Antibodies were diluted 1:100 to 1:200, and samples were incubated overnight at 4°C in a dark room. After washing with Tris-buffered saline supplemented with Tween 20 (TBST), each section was exposed to secondary antibodies for 1 hour. After another wash with TBST, the sections were exposed to 4',6-diamidino-2-phenylindole (PureBlu; Bio-Rad, Hercules, CA, USA). Fluorescence microscopy and/or confocal microscopy (Axio Imager 2; Carl Zeiss, Oberkochen, Germany) were used to examine the samples.