Horseradish peroxidase avidin d

Purified Hp of 60 μl of sera (pooled from 20 patients, 3 μl per patient) from LC and HCC, respectively, was analyzed by lectin blot according to the previous description (Shu et al., 2011 (link)). Five biotinylated lectins (Vector Laboratories, Burlingame, CA) including Amaranthus caudatus lectin (ACA, 1 μg/ml), Griffonia simplicifolia lectin I (GSI-L, 1 μg/ml), Jacalin (JAC, 1 μg/ml), Vicia villosa lectin (VVA, 1 μg/ml), and Wisteria floribunda lectin (WFA, 2 μg/ml) were incubated for 45 min, respectively. After washing and incubating with horseradish peroxidase Avidin D (Vector Laboratories, Burlingame, CA), the bands were developed using chemiluminescence detection reagents (GE Healthcare, Piscataway, NJ).

We have published the B16-OVA tumor model previously [57 (link)]. In brief, B16-OVA was a gift from Dr. Economou, University of California at Los Angeles and cultured in RPMI-1640 media (Mediatech, Hernden, VA, USA) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 10,000 IU penicillin, 10,000 μg/mL streptomycin, 25 μg/mL amphotericin (Mediatech), 0.05 mM 2-mercaptoethanol (Sigma), and kept under 0.4 mg/mL G418 selection (Research Products International Corp., Mt. Prospect, IL, USA). A vaccine of B16-OVA cells was made by lethally irradiating them (25 Gy) and injecting 10⁷ cells, or saline, i.p. into Nrf2–/– or WT mice. Seven days later, spleens were harvested and OVA-specific IL-4 and IFN-γ producing T cells enumerated by ELISPOT assay by plating 2 × 10⁵/well for 48 h in anti-IL4- or anti-IFNγ-coated (BD Pharmingen, Franklin Lakes, NJ, USA) MultiScreen-HA plates (Millipore Corp, Billerica, MA, USA). Cells secreting IFNγ or IL-4 were detected using the corresponding biotinylated anti-IL-4 or anti-IFNγ antibodies (BD Pharmingen) and 200× diluted horseradish peroxidase avidin D (Vector Laboratories, Burlingame, CA, USA), with spots being developed in the presence of 0.4 mg/mL 3-amino-9-ethyla-carbazole substrate (AEC, Sigma) and counted using an ImmunoSpot Image Analyzer (Cellular Technology Ltd., Cleveland, OH, USA).

Cortical tissues were homogenized with a Polytron homogenizer in ice cold TBS buffer containing protease inhibitor and phosphate inhibitor cocktails (Roche). The homogenates were centrifuged at 100,000 g for 30 min at 4°C and the supernatants were collected. ApoE levels were analyzed using an ELISA. Briefly, 96-well plates were coated overnight with an apoE antibody (AB947, Millipore) in carbonate buffer at 4°C overnight. The plates were blocked with 1% Block Ace in PBS, and then washed with PBS 3 times. Recombinant apoE (Fitzgerald) along with samples were diluted and added at a volume of 100 μl/well incubated at 4°C overnight. The plates were washed and incubated with biotin-conjugated goat anti-apoE antibody (Meridian Life Science) for 2 h at room temperature. After incubation with Horseradish Peroxidase Avidin D (Vector Laboratories) for 90 min at room temperature, the plate was developed by adding tetramethylbenzidine Super Slow substrate (Sigma). The reaction was stopped and read at 450 nm with a microplate reader.

Immunohistochemical staining of CGRP was performed with the avidin-biotin method with a protocol similar to that for c-Fos staining except using a 30 min-blocking incubation, anti-CGRP rabbit polyclonal antibody (1:1000, Calbiochem, San Diego, CA, United States), and horseradish peroxidase avidin D (1:200 Vector Labs, Burlingame, CA, United States).