Triplestain ihc kit

Immunohistochemical staining was performed on the paraffin-embedded tissue sections. Endogenous peroxidase was blocked with 3% hydrogen peroxide, and then with 5% animal serum for 30 min to block non-specific reactions. These tissue sections were incubated with primary antibodies (see antibody list in Additional file 1). Tissue sections were incubated with horseradish peroxidase-conjugated secondary antibodies (1, 300–400 dilutions with PBS) for 2 h at room temperature, and then incubated with peroxidase substrate DAB kit (Vector Laboratories, Inc., Burlingame, CA) to develop brown color. The counterstaining was performed using hematoxylin or methyl green. A negative controls without primary antibodies was included in each run. Triple staining was performed on human sample using a Triplestain IHC Kit (Abcam, ab183286, Cambridge, MA) according to the provided factory instructions. Digital images were acquired with the Olympus 1 × 51 microscope (Olympus, Pittsburgh, PA) at 10× magnification using the Olympus DP72 digital camera and the length of scratch-wound was measured via the cellSens Dimention imaging system.

All human specimens and all experimental protocols were studied after approval by the Kagawa University and University of Chicago Institutional Review Board. Informed consent was obtained from all subjects. All methods were carried out in accordance with relevant guidelines and regulations. Formalin-fixed paraffin-embedded tissue blocks were obtained from the tissue bank archives at the Kagawa University. AD and psoriasis specimens were used for immunohistochemical analysis of CD68, CD206 and iNOS protein expression. TripleStain IHC kit (Abcam) was utilized to examine the colocalization of CD206 and iNOS protein.