Vector vip peroxidase

Free-floating immunohistochemistry for tyrosine hydroxylase (TH) was used to visualize norepinephrine cells in LC from a parallel series of sections. Sections were blocked in 10% goat serum in Tris-buffered saline with Tween-20 (TBS-T) solution for 1 h and incubated with rabbit anti-TH primary antibody (1:1000; P40101; Pel-Freez Biologicals, Rogers, AR, USA) diluted in 3% goat serum TBS-T for 48 h at 4 °C. Sections were then incubated with a biotinylated goat anti-rabbit antibody (1:200; BA-1000; Vector Laboratories, Burlingame, CA, USA) with 3% goat serum in TBS for 1 h, and then in Vectastain ABC 1:500 in TBS-T (SK-4005) for 1 h. Staining was visualized with Vector VIP peroxidase for 10 min (SK-4600; Vector Laboratories). The sections were mounted on glass slides, dried, dehydrated in alcohol, defatted in xylene and cover slipped using DPX mounting media.

The tissues for histology were fixed with 10% formalin, dehydrated through a graded series of alcohol washes, and embedded in paraffin. Paraffin sections were stained with safranin-O for GAG detection, and Fast Green was used as a counterstain. For immunohistochemistry, slides were deparaffinized, hydrated, and antigen retrieval performed by incubation with pronase (1 mg/ml in PBS containing 5 mM calcium chloride; Sigma-Aldrich) for 10 min at room temperature. Sections were then incubated with PBS/BSA for 10 min at room temperature, then overnight at 4 °C with primary antibody (mouse anti-type II collagen – DSHB II-II6B3, 1/500 in PBS/BSA, mouse anti-typeX collagen – kind gift of Gary Gibson (Henry Ford Hospital, Detroit, MI) - 1/500 in PBS/BSA, mouse anti-elastin – millipore MAB2503, 1/200 in PBS/BSA). After 3 washes, secondary antibody (biotin conjugated goat anti-mouse; Cappel 1/200 in PBS/BSA) was added and sections incubated for 1 h at room temperature. After 3 washes, sections were incubated with streptavidin-HRP (Gibco, 1/200 in PBS/BSA) at room temperature for 30 min. Slides were washed 3 times and detection performed using Vector VIP peroxidase (SK-4600; Vector Labs). Slides were then counterstained with Fast Green (0.0125% in 25 % ethanol, 0.875 % glacial acetic acid) 10 s, then dehydrated and mounted with Permount (Fisher Scientific).