Vector dab peroxidase hrp substrate kit

Tissue-section slides were de-paraffinized in xylene and rehydrated in ethanol, followed by antigen retrieval with 10% citrate buffer (pH 6.0) in a pressure cooker for 10 minutes at 120 °C. Slides were incubated with the primary antibody at 4°C overnight. For RARRES2 staining, tissues were fixed with 10% formalin and embedded in paraffin. Anti-chemerin antibody (Abcam, Cambridge, MA, ab72965) was used at 12.5 μg/ml. Immunostaining was performed using the Dako Envison+ System-HRP (DAB) for mouse primary antibodies (Dako, Carpinteria, CA). IHC slides were scored using the formula Total staining intensity = Σ (intensity scores × percent positivity), where intensity score 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining. Interpreter of the IHC was blind to the diagnosis and gene expression levels accessed by real-time qPCR. For phospho-p38 staining, primary antibody used was phospho-p38 (Thr180/Tyr182) antibody (1: 800, Cell Signaling, Danvers, MA, #4511). Immunostaining was performed by detection with SignalStain Boost IHC detection reagents (HRP, Cell signaling, rabbit #8114), followed by staining with Vector DAB peroxidase (HRP) substrate kit (Vector laboratories, Burlingame, CA, SK-4100). Hematoxylin (Sigma-Aldrich, St. Louis, MO) was used for counterstaining.

Antigens were detected in paraffin-embedded tissues using the VECTASTAIN® Elite ABC-HRP Kit (Vector® Laboratories, Burlingame, CA) and the Vector DAB Peroxidase (HRP) Substrate Kit with standard protocols. Antigen retrieval was performed by immersing the slides in citrate buffer (0.01 M, pH 6.0) preheated via microwave oven for 45 s, followed by heating for 3 × 5 min at 50% power, replenishing the buffer as needed between intervals. Tissue sections were blocked with 1× protein blocking solution (Vector Animal-free Blocker) and normal goat serum (1:50 dilution) for 60 min. Primary antibodies were diluted with the protein blocking solution and incubated overnight at 4°C in a hydration chamber. Antibodies included: anti-smooth muscle α-actin (D-SMA, rabbit polyclonal, 1:400; Millipore, Temecula, CA), and macrophage marker F4–80 (1:20; Abcam®, San Francisco, CA). Secondary antibodies were diluted 1:50 in protein blocker plus appropriate serum, and incubation was performed for 60 min at room temperature. The slides were briefly counterstained with Gill’s 3 hematoxylin, dehydrated through graded alcohols to xylene and coverslipped with a permanent mounting medium prior to imaging.