Increased caveolin-1 in intervertebral disc degeneration facilitates repair

Table 2 Details of the immunofluorescence protocol

Antibody	Specifications	Host	Dilution	Secondary antibody
Tie2	324 (Santa Cruz Biotechnology)	Rabbit	4 μg/ml	Alexa Fluor® 594 AffiniPure Donkey Anti-Rabbit IgG (711-165-152; Jackson ImmunoResearch, West Grove, PA, USA)
GD2	554272 (BD Biosciences)	Mouse	10 μg/ml	Alexa Fluor™ 488 Donkey Anti-Mouse IgG (A21202; Molecular Probes/Invitrogen, Eugene, OR, USA)
Caveolin-1	2910 (Abcam)	Rabbit	5 μg/ml	Alexa Fluor® 594 AffiniPure Donkey Anti-Rabbit IgG (711-165-152; Jackson ImmunoResearch)
pSMAD2	3101 s (Cell Signaling Technology, Danvers, MA, USA)	Rabbit	5 μg/ml	Alexa Fluor™ 488 Donkey Anti-Rabbit IgG (A21206; Molecular Probes/Invitrogen)
Negative control	Primary antibody omitted	–	–	Alexa Fluor™ 488 Donkey Anti-Mouse IgG (A21202; Molecular Probes/Invitrogen) + Alexa Fluor® 594 AffiniPure Donkey Anti-Rabbit IgG (711-165-152; Jackson ImmunoResearch)

GD2 disialoganglioside 2, IgG immunoglobulin G, pSmad2 phosphorylated Smad2
The thoracic intervertebral discs were embedded in O.C.T. compound (14020108926; Leica Microsystems), transverse 10-μm cryosections were mounted on Superfrost Plus slides (4951PLUS4; Thermo Scientific), and the sections were treated with phosphate-buffered saline (PBS) diluted 1:1 in Gibco TrypLE™ Express Enzyme (12605; Life Technologies, Carlsbad, CA, USA) for 10 minutes at 37 °C. The sections were blocked with 3 % bovine serum albumin (BSA) and 0.25 % Triton X-100 (T8787; Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes and incubated overnight at 4 °C with the primary antibodies diluted in 1 % BSA in PBS. The secondary antibody was diluted 1:250 in 1 % BSA and 0.1 % Triton X-100 in PBS and applied for 45 minutes at room temperature. The sections were mounted with VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (H-1200; Vector Laboratories)

ISSN: 1478-6362