Superblock t20 pbs blocking buffer

Cell lysates (10 μg) or purified podoplanin (0.1 μg) were boiled in SDS sample buffer (Nacalai Tesque, Inc., Kyoto, Japan)26 (link). The proteins were electrophoresed on 5–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd.) and were transferred onto a PVDF membrane (EMD Millipore Corp., Billerica, MA). After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the membrane was incubated with primary antibodies or biotinylated lectin (1 μg/ml; Vector Laboratories Inc., Peterborough, UK), then with peroxidase-conjugated secondary antibodies (Dako; 1/1,000 diluted) or streptavidin-HRP (Dako; 1/1,000 diluted), and developed with the ECL-plus reagent (Thermo Fisher Scientific Inc.) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Peptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific) at 1 μg/ml. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific), the plates were incubated with culture supernatant with subsequent 1:2000 diluted peroxidase-conjugated anti-mouse IgG (Dako). The enzymatic reaction was conducted with 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories). These reactions were performed with a volume of 50–100 μl at 37 °C.

Synthesized ATRX peptides using PEPScreen (Sigma-Aldrich Corp., St. Louis, MO, USA) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 10 μg/mL for 30 min at 37 °C. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the plates were incubated with 10 μg/mL purified AMab-6 followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies Inc.). The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). Optical density was measured at 655 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). These reactions were performed at 37 °C using a total sample volume of 50–100 μL.

Enzyme-linked immunosorbent assay was performed as previously described.^{61 (link)} Briefly, 96-well enzyme-linked immunosorbent assay plates (Nalge Nunc International, Rochester, NY, USA) were coated with 1 μg/ml of capture antibody (Ab62) in 50 mM carbonate buffer (pH 9.6) at 4 °C overnight. After washing with PBS with 0.05% Tween 20 (PBST), SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Rockford, IL, USA) was added to each well. After incubation for 1 h at room temperature with shaking, plates were washed five times in PBST. Samples and standards were incubated at room temperature for 2.5 h with shaking. After washing with PBST, 1 μg/ml of biotinylated Ab62 in blocking buffer was added to each well. After incubation for 1.5 h at room temperature, the plates were washed with PBST. Avidin-conjugated peroxidase (ExtrAvidin, Sigma) was added to each well. After incubation for 1 h at room temperature, plates were washed with PBST. One hundred microliters of 3,3′,5,5′-tetramethylbenzidine solution (Sigma) were added to each well and incubated for 15 min with shaking. To stop the reaction, 50 μl of 2 N H₂SO₄ was added to each well. The absorbance was measured at 450 nm.

The virucidal efficacy of the Au/CuS NPs was evaluated using an ELISA method previously reported by our group [51 ]. Briefly, 50.0 μL of untreated or treated VLP solution was dispersed into the wells of a medium-binding 96-well polystyrene plate (Costar^™ 3591; Corning Incorporated, Corning, NY) and incubated at room temperature for 1 h. 50.0 μL of 0.01M PBS was used as a blank. Each well was washed with 0.01 M PBS and blocked with 100.0 μL of SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific). The wells were washed with PBS and sequentially treated with 0.2 μg/mL mAb 3901 anti-GI.1 VLP and 0.1 μg/mL HRP-labeled goat anti-mouse IgG antibody solutions for 1 h. The plate was washed twice with 100.0 μL aliquots of 0.01 M PBS + 0.05% Tween^™ 20 between steps. Following the final washing step, each well was reacted with 100 μL of TMB (3,3’,5,5’-tetramethylbenzidine) Peroxidase Substrate Microwell Substrate System (KPL, Gaithersburg, MD) for 10 min, filled with 50.0 μL of Stop Solution (KPL, Gaithersburg, MD), and the absorbance of each well was read at 450 nm using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). Reduced absorbance in NP-treated samples (compared to an untreated control) indicated reduced concentration of intact VLPs due to damage to the capsid surface proteins and associated VLP inactivation.

One formalin-fixed paraffin-embedded (FFPE) tissue sample from an oropharyngeal squamous cell carcinoma patient who underwent surgery at Sendai Medical Center was used for this study (13 (link)). Written informed consent was obtained from the patient for sample procurement and subsequent data analyses. Tissue microarray (CC00-10-001) including lymphomas, normal lymph node, and normal thyroid was purchased from Cybrdi, Inc.
The 4-µm thick paraffin-embedded tissue sections were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with the SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.), tissue sections were incubated with C₂₀Mab-11 (5 µg/ml) for 1 h at room temperature and treated with the Envision+ Kit for mouse (Agilent Technologies, Inc.) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Synthesized rPDPN peptides using PEPscreen (Sigma-Aldrich Corp., St. Louis, MO) were immobilized on Nunc MaxiSorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at 10 μg/mL for 30 minutes at 37°C. After blocking with Superblock T20 (PBS) blocking buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with purified PMab-2 (10 μg/mL), followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies, Inc.). The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). Optical density was measured at 655 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions were performed at 37°C with a total sample volume of 50–100 μL.