Increased caveolin-1 in intervertebral disc degeneration facilitates repair

Table 1 Details of the immunohistochemistry protocol

Target protein	Clone/catalog number (manufacturer)	Origin	Antigen retrieval	Block	Concentration of first Ab	Second Ab (catalog number; manufacturer)
Caveolin-1	Clone 2297, 610406 (BD Biosciences, San Jose, CA, USA)	Mouse	Citrate (10 mM), 70 °C, 60 minutes	0.3 % H₂O₂ + 10 % goat serum	5 μg/ml in PBS	Goat anti-mouse, (K4001; Dako, Carpinteria, CA, USA)
Ki-67	Clone SP6, RM-9106-S (Thermo Scientific™, Waltham, MA, USA)	Rabbit	Citrate (10 mM), 80 °C, 90 minutes	0.3 % H₂O₂ + 10 % goat serum	4 μg/ml in PBS	Goat anti-rabbit, (K4003; Dako)
Collagen type II	II-II6B3 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA)	Mouse	1 mg/ml pronase + 10 mg/ml hyaluronidase, 37 °C, 30 minutes	0.3 % H₂O₂ + 5 % BSA/PBS	0.02 μg/ml (murine, canine) or 0.4 μg/ml (human) in 5 % BSA/PBS	Goat anti-mouse, (K4001; Dako)
Collagen type I	ab6308 (Abcam, Cambridge, UK)	Mouse	1 mg/ml pronase + 10 mg/ml hyaluronidase, 37 °C, 30 minutes	0.3 % H₂O₂ + 5 % BSA/PBS	0.1 μg/ml (human) or 0.07 μg/ml (canine) in 5 % BSA/PBS	Goat anti-mouse, (K4001; Dako)

Ab antibody, BSA bovine serum albumin, PBS phosphate-buffered saline
Midsagittal intervertebral disc sections were deparaffinized with xylene and graded ethanol. The primary antibody was applied at 4 °C overnight. In control experiments, the primary antibody was replaced with mouse immunoglobulin G1 (3877; Santa Cruz Biotechnology), and no false-positive staining was observed. The secondary antibody was applied for 60 minutes, and the sections were incubated with a liquid 3,3′-diaminobenzidinesubstrate chromogen system (K3468; Dako) for 10 minutes. The slides were stained with Vector Hematoxylin QS solution (H3404; Vector Laboratories, Burlingame, CA, USA) for 10 seconds, dehydrated, and mounted (H5000; Vector Laboratories)

ISSN: 1478-6362