Biotinylated anti rabbit immunoglobulin g

After the brain sections were deparaffinized and rehydrated, they were boiled in citrate buffer (pH 6.0) for antigen recovery, incubated with 3% hydrogen peroxide (H₂O₂) for 20 min, and then 3% bovine serum albumin in PBS for 60 min (to block non-specific binding). After pretreatment, the sections were incubated overnight with rabbit anti-mouse MPO antibodies (dilution 1:100, Abcam, Cat #: ab9535), rabbit anti-mouse Iba-1 antibodies (dilution 1:500, Wako, Cat #: 019-19741), and goat anti-arginase 1 (dilution 1:100, Santa Cruz Biotechnology, Cat #: sc-18355). They were then incubated with biotinylated anti-rabbit immunoglobulin G (dilution 1:400, Vector) for 2 h at RT. The bound antibody was recognized using an avidin-peroxidase conjugate solution (ABC, 1:1000; Vector) and visualized using diaminobenzidine. The negative control slices were treated with the same immunoblotting procedures but without the primary antibody.

Immunostaining with antibodies for fibrillin-1 (rabbit polyclonal, 1:300; Bioss Antibodies, Inc., Woburn, MA) and fibrillin-2 (rabbit polyclonal, 1:300; Bioss Antibodies) was performed in 3 eyes of 3 patients with MH (Cases 1–3) and in 1 eye of 1 patient with ERM (Case 4). After collection, the BPM and VC specimens were fixed with 4% paraformaldehyde, rinsed with phosphate buffer saline (PBS, pH 7.4), and blocked with 5% normal goat serum. Immunostaining was then performed by the avidin–biotin complex method with each target antibody. Next, the BPM and VC specimens were incubated with antibodies for fibrillin-1 and -2 at 4 °C for 2 days, rinsed with PBS, and incubated with biotinylated anti-rabbit immunoglobulin G (1:1000; Vector Laboratories, Inc., Burlingame, CA) at room temperature for 2 h. After a rinse with PBS, the specimens were incubated with alkaline phosphatase-labeled avidin–biotin complex (Vector Laboratories) at room temperature for 2 h and treated with an ImmPACT™ Vector^® Red alkaline phosphatase substrate (Vector Laboratories). After dehydration, the specimens were enclosed with Entellan^® new (Merck, KGaA, Darmstadt, Germany) and observed under a BZ-x700 microscope (Keyence Corporation, Osaka, Japan).