Detection of immunostaining was performed using a Dako Rabbit Envision HRP polymer kit (Dako, Cat. #51699).
The IL-6 and NF-κB samples were visualized using a Novo Link Polymer Kit (RE7280-K, Leica Microsystems, Tokyo, Japan).
Then, a NovaRED horseradish peroxidase (HRP) kit (Vector) was used for PDE10A staining, and a ZymedChem-plus alkaline phosphatase (AP) Polymer kit (Zymed) was used for PCNA staining according to the manufacturer's instructions.
Slides were quenched in 3% hydrogen peroxide, steamed for antigen retrieval in DIVA/citrate buffer (pH 6.0) (BIOCARE ref; DV2004MX) for 20 minutes, and incubated in Rodent block (BIOCARE MM HRP- Polymer Kit ref: MM510G) for 30 minutes.
Double immunohistochemical staining was performed with a Polymer kit (GBI, Seattle, WA, USA).
Staining was completed with Novolink Max DAB (Polymer) Kit (Leica Biosystems, Wetzlar, Germany).
Most detection was performed with the Novocastra Novolink Polymer Kit (Leica Microsystems, Buffalo Grove, IL).
For blocking and detection, a HRP-conjugated polymer kit according to the manufacturer's instructions (Zytomed Systems) was used.
Peroxidase conjugation was performed using the Envision + HRP labeled Polymer Kit from Dako, and diaminobenzidine (DAB+, Dako) was the chromogen.
The detection kits ZytoChem Plus HRP Kit, anti-Rabbit and ZytoChem Plus (HRP) Polymer Kit, anti-Mouse (Zytomed Systems, Berlin, Germany) were utilized following the manufacturer's instructions.
