Alexa488 and alexa594 conjugated antibodies

Mouse tail cross sections (4 to 5 mm thick) were fixed for 2 hours at 4 C. Samples were then permeabilized with 0.5% Triton X-100 in PBS for 2 days, washed, and blocked with PermBlock solution (1% bovine serum albumin, 0.1% Tween 20) for 2 days (room temperature). Rabbit antie LYVE-1 (AngioBio) and rat anti-CD31 antibodies were used for staining, by incubation in PermBlock solution for 7 days at 4 C under constant agitation. After multiple washing steps in PBS with 0.1% Tween 20 for 2 days, the samples were incubated for 7 days in PermBlock with Alexa488-and Alexa594-conjugated antibodies at 4 C (Invitrogen). Tissue samples were then embedded in 1% ultrapure agarose gels on ice and dehydrated in a series of methanol solutions (50%, 70%, 95%, 100%), each step for more than an hour with the last step overnight. Tissue clearing was achieved with incubation in 50% benzyl alcohol/benzyl benzoate (Acros Organics, Thermo Fischer; 1:2) for 3 to 4 days, until the tissue was visibly clear. Samples were stored in 50% benzyl alcohol/benzyl benzoate at 4 C in the dark. Whole mount image acquisition was performed using an Olympus MVX10 LaVision BioTec Ultramicroscope (LaVision, Bielefeld, Germany), and images were processed using Imaris 7.6.4 (Bitplane).

Each ERM specimen was collected during vitrectomy surgery for ERM. The tissue was immediately fixed with 4% paraformaldehyde and cryoprotected; then, 10-lm sections were obtained, as previously described. 33, 34 The sections were stained with hematoxylin and eosin, or immunostained with anti-alpha smooth muscle actin (aSMA) antibody (1:200; Sigma-Aldrich, St. Louis, MO, USA), anti-glial fibrillary acidic protein (GFAP) antibody (1:400; Cell Signaling Technology, Danvers, MA, USA), and anti-collagen type I antibody (1:200; Rockland Immunochemicals, Inc., Limerick, PA, USA); they were then visualized with Alexa 488-and Alexa 594-conjugated antibodies (1:1000; Invitrogen, Carlsbad, CA, USA), as well as 4 0 , 6diamidino-2-phenylindole (DAPI; Invitrogen). Images were acquired with a BioImaging Navigator fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). This study was conducted in accordance with the guidelines of the Declaration of Helsinki; the protocol was registered within the UMIN Clinical Trial Registry (registered number UMIN000024553) and approved by the Nagoya University Hospital Ethics Review Board. Written informed consent was obtained from all participating patients.