Rabbit monoclonal anti phospho erk1 2

Immunohistochemical staining was performed according to our routine protocol using an avidin-biotin-peroxidase method (16 (link)). The primary antibodies included rabbit polyclonal anti-myeloperoxidase (MPO) (Abcam, Cambridge, MA, USA.), rat monoclonal antibody to murine MAC3 (Novus Biologicals, Littleton, CO, USA), mouse monoclonal anti-proliferating cell nuclear antigen (PCNA) (Calbiochem, Gibbstown, NJ, USA), goat polyclonal anti-cytokeratin 19 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-amylase (Santa Cruz Biotechnology), and rabbit monoclonal anti-phospho-ERK1/2 (Cell Signaling Technology, Boston, MA, USA) antibody. The appropriate biotinylated secondary antibody and avidin-biotin-peroxidase complex (Vector Lab, Burlingame, CA, USA) were used for detecting antigen–primary antibody complex. A characteristic brown color was developed by incubation with 3,3-diaminobenzidine substrate chromogen system (Sigma-Aldrich, St. Louis, MO, USA). The negative control was established by replacement of primary antibody with normal serum, and an appropriate positive control was used for each primary antibody. Specific antibody-labeled signals were analyzed under a Nikon research microscope (Nikon instrument Inc., Melville, NY, USA) for positive cell number and staining intensity.

All reagents were purchased from Sigma (St. Louis, MO) unless noted otherwise. Anti-MUC1 mouse monoclonal antibody (GP1.4, Cat. # MA5–13168), Alexa Fluor 488-conjugated rabbit anti-mouse or goat anti-rabbit IgG, and Alexa Fluor 594-conjugated rabbit anti-mouse or goat anti-rabbit IgG were from Thermo Fisher Scientific (Waltham, MA). Polyclonal rabbit anti-mouse MUC1 antibody was from Abcam (Cat. # ab15481, Cambridge, MA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit IgG antibodies were from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA). The pCI-MUC1 expression plasmid and pCI-neo empty vector were a generous gift from Dr. Sandra J. Gendler (Mayo Clinic, Scottsdale, AZ). MUC1 smart pool siRNA and non-targeting control siRNA were purchased from Dharmacon (Lafayette, CO). Rabbit monoclonal anti-total ERK1/2, rabbit monoclonal anti-phospho-ERK1/2, and HRP-conjugated mouse monoclonal anti-beta actin antibodies were purchased from Cell Signaling (Danvers, MA). Polyclonal mouse anti-major surface glycoprotein (Msg) of Pneumocystis murina (P. murina) was generated as previously described (Bishop, Helman, & Kovacs, 2012 (link)). Human IL-6 and IL-8 ELISA kits were from R&D (Minneapolis, MN).