Envison system hrp dab for mouse primary antibodies

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.