Hrp conjugated streptavidin

MDA cells undergoing different treatments were plated at similar cell numbers, and 48-96 h later, cell supernatants were collected. When relevant, one day after cell plating, the cells were exposed to kifunensine or its vehicle control for 72 h, followed by exposure to PD-1 or its control for additional 72 h, ending with collection of cell supernatants.
CXCL8 levels were determined in cleared supernatants (by centrifugation) and ELISA analyses were performed, using the following antibodies and recombinant proteins (all from Peprotech, Rocky Hill, NJ, USA): CXCL8 coating antibodies: #500-P28; CXCL8 detecting antibodies: #500-P28BT; recombinant human CXCL8: #200-08. HRP-conjugated streptavidin (#016-030-084, Jackson Immunoresearch laboratories) and substrate TMB/E solution (#ES001, Millipore, Burlington, MA, USA) were added, the reaction was stopped by addition of 0.18 M H2SO4 and absorbance was measured at 450 nm.

MOPC-21 (IgG₁), pFLAG-CTC vector, and Streptavidin-FITC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD44 (clone 156-3C11) and anti-α2 integrin subunit (clone P1E6, IgG1) antibodies were purchased from Millipore (Billerica, MA, USA). FITC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-rabbit IgG, HRP-conjugated rabbit anti-mouse IgG, HRP-conjugated rat anti-rabbit IgG, and HRP-conjugated Streptavidin were purchased from Jackson Immunoresearch (West Glove, PA, USA). Anti-CD44v10 antibody (AB2082) was purchased from Calbiochem (San Diego, CA, USA). Anti-FLAG (M2) antibody was purchased from Stratagene (Santa Clara, CA, USA). Type I Collagen was purchased from Inamed Biomatenak (Freemont, CA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from GE Healthcare (Piscataway, NJ, USA). Other chemicals were purchased from Sigma-Aldrich unless otherwise mentioned.

P particle binding to HBGAs was measured by saliva- and synthetic oligosaccharide-based binding assays. The saliva samples were selected from a previously described panel of saliva with well-defined A, B, O, and Lewis antigens [15 (link),35 (link)]. Boiled saliva samples were diluted 1:1000 with 1XPBS and coated onto 96-well microtiter plates at 4°C overnight. After blocking with 5% nonfat dry milk, P particles (20 μg/ml) were added after 3-fold serial dilution and incubated at 37°C for 1 hr. The bound P particles were detected using guinea pig anti-NoV (1:3330) sera, followed by HRP-conjugated goat anti-guinea pig IgG (ICN, Aurora, OH). The signals were developed using a TMB substrate kit.
For the oligosaccharide-based binding assays, microtiter plates were coated with recombinant P particles (2 μg/ml) at 4°C overnight. After blocking with 5% Blotto nonfat milk, oligosaccharide-polyacrylamide (PAA)-biotin conjugates (2 μg/ml) were added and incubated at 4°C overnight. Oligosaccharides used in this study included H type 1, H type 2, H type 3, type A and type B disaccharides, type A and type B trisaccharides, Le^a, Le^x, Le^b, Le^y, and sialic-Le^a and sialic-Le^x tetrasaccharides (GlycoTech Corporation, Rockville, MD). Bound oligosaccharides were detected by HRP-conjugated streptavidin (Jackson Immuno Research Laboratories Inc., West Grove, PA).

Cytokines were quantified from supernatants of stimulated cells. IL-10 and IL-12p40 were quantified by ELISA. Cone JES5-2A5 (eBioscience) was used for IL-10 capture, and biotinylated anti-mouse IL-10 SXC-1 (BD Biosciences) was used for detection. Clone C15.6.7 was used for IL-12p40 capture, and biotinylated anti-mouse IL-12p40 C17.8 was used for detection (both gifts from DNAX Research Institute), followed by HRP-conjugated streptavidin (Jackson ImmunoResearch Laboratories). IL-12p70, TNF-α, and IL-27 (eBioscience), IL-1β (R&D Systems), and IFN-β (PBL) were quantified using commercially available ELISA kits.

4T1 tumors and dLNs were harvested on indicated days, fixed in freshly prepared 4% paraformaldehyde for 1 hr at 4°C and incubated overnight in 30% sucrose. Tumors were then frozen in optimum cutting temperature (OCT) medium and stored at −80°C. Sections (5 μm) were treated with 3% H₂O₂ in PBS for 30 minutes in order to eliminate endogenous peroxide activity. After blocking in 4% hamster serum for an hour and three washes with PBS, anti-mouse CD11c eFluor 615 (eBioscience, San Diego, CA) was applied at a 1/100 dilution in 1% BSA for 1 hour, followed by three washes and incubation in HRP-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) at a 1/2,000 dilution in 1% BSA for 30 minutes. To amplify the signal, slides were subject to two rounds of biotin-tyramide amplification (PerkinElmer, Melville, NY) using manufacturer’s suggested protocol. The slides were counterstained with DAPI, mounted with Vectashield and images obtained using a Nikon Eclipse 800 deconvolution microscope. Positive cells were counted from at least 3 randomly selected fields (20× magnification).