Streptavidin biotin blocking solution

All tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin, and sections (5-µm thickness) were stained with hematoxylin and eosin or processed for antibody (Ab) staining. For IHC staining, sections were deparaffinized and treated with an antigen-retrieval solution (Dako, Carpinteria, CA) at 98°C for 25 min, a peroxidase-blocking solution (Dako) for 15 min, and then with a streptavidin-biotin blocking solution (Vector Laboratories, Burlingame, CA) for 1 h. Sections were incubated for 2 h at room temperature with one of the following antibodies: rabbit anti-O. tsutsugamushi Karp strain polyclonal antibody (pAb, 1∶500); rat anti-mouse neutrophil monoclonal antibody (mAb, 1∶25, clone 7/4, Caltag Laboratories, Buckingham, UK); rabbit anti- myeloperoxidase (MPO) pAb (1∶25, Abcam); or rabbit anti-mouse CD3 mAb (1∶25, Abcam, Cambridge, MA). Biotinylated goat anti-rat or anti-rabbit secondary antibodies (1∶200, Vector) were incubated on the sections for 30 min. Sections were stained with alkaline phosphatase-conjugated streptavidin (1∶200, Vector), Vector Red alkaline phosphatase substrate, and counterstained with hematoxylin (Sigma, St. Louis, MO). Reagent negative controls consisted of samples in which primary Ab was replaced with normal rat or rabbit IgG. Sections were dehydrated, mounted in Permount (Vector), and imaged under an Olympus BX53 microscope.