Kpl sureblue tmb microwell peroxidase substrate

Peroxidase conjugated ABC reagent, Vectastain Elite ABC standard kit was from Vecter Laboratories, Inc. (Burlingame, CA, USA). Antihuman AGP rabbit serum and Protein Block Serum-Free Reagent were from Dako (Carpinteria, CA, USA). Peroxidase conjugated anti-human AGP and Universal HIER antigen retrieval reagent were from Abcam (Cambridge, UK). KPL SureBlue TMB Microwell Peroxidase Substrate was from Sera Care Life Sciences (Milford, MA, USA). Anti-PD-L1 and SignalStain Boost IHC Detection Reagent were obtained from Cell Signaling Technology (Danvers, MA, USA). N-Histofine High Stain HRP (Multi) was from Nichirei Biosciences Inc. (Tokyo, Japan). PNGase F was from Roche Applied Science (Indianapolis, USA). BlotGlyco was obtained from Sumitomo Bakelite, Co. (Tokyo, Japan). Neuraminidase (Arthrobacter ureafaciens, 1U/ml) was purchased from Nacalai Tesque (Kyoto, Japan). Biotinylated Aleuria aurantia lectin (AAL) was kindly provided by Prof. Naohisa Kochibe, Gunma University. Tumor-associated antigens in serum samples measured in this study were carcinoembryonic antigen (CEA) and squamous cell carcinoma (SCC). Levels of each antigen were determined by an ELISA using the Cobas system (Roche for CEA) and the ARCHITECT system (Abbot for SCC) and standard cut-off values were set at 5.0 ng/ml for CEA and 1.5 ng/ml for SCC, respectively.

100 μL BALF of each mouse was quadruple added into the 96-well plates, mixed with 100 μL coating buffer, and then incubated at 4 °C overnight. After washing three times with 1x TBST, the pre-coating plate was subsequently blocked with 5% fat-free in 1xTBST at room temperature for 1 h, washed with 1xTBST, incubated with rabbit anti-IL-1β mAb (ab9722; Abcam) for 1 h, and reacted with HRP-conjugated goat anti-rabbit IgG antibody. Finally, the immune-reactive complexes were reacted with KPL SureBlue™ TMB Microwell Peroxidase Substrate (SeraCare, West Bridgewater, MA, USA) plus the stop buffer (1N HCl), and recorded by measuring the optical absorbance at 450 nm using the ELISA plate reader SpectraMax iD3 (MOLECULAR DEVICES, Wals, Austria).