ELISPOT assays were performed as previously reported22 (link). Millicell membranes, on which cells were cultured with M-CnT for 12 h, were washed three times with PBST and blocked with 1% BSA for one hour at 37°C. After washing with PBST, the Millicell wells were pre-treated with a VECTASTAIN ABC kit (Vector Lab., Burlingame, CA) and 10% H2O2, then subsequently incubated overnight with an anti-Wnt-7a antibody (clone E-9, diluted 1:100, Santa Cruz) at 4 °C. After washing with PBS, the wells were incubated with a biotinylated anti-mouse IgG antibody (1:500; Santa Cruz Biotech) and exposed to a VECTASTAIN ABC kit. Spots were visualized by addition of True Blue™ (SeraCare Life Sciences, Inc.) as a substrate solution. The number of spots in each well was then counted using a stereo dissecting microscope.
Vectastain abc kit
Manufactured by Santa Cruz Biotechnology
The VECTASTAIN ABC kit is a reagent system used for the detection and visualization of antigens in immunohistochemical and immunocytochemical procedures. The kit contains the necessary components to perform an avidin-biotin complex (ABC) immunodetection method.
Automatically generated - may contain errors
2 protocols using vectastain abc kit
1
Quantifying Wnt-7a Expression using ELISPOT Assay
2
Immunohistochemical Analysis of ER-α and ER-β
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The para n sections were dewaxed by routine method and immersed in sodium citrate antigen retrieval solution (pH 6.0) for complete antigen retrieval. Thereafter, they were incubated for 25 min with 3% hydrogen peroxide (H 2 O 2 ). Each section was incubated with blocking serum (Vectastain ABC kit) at room temperature for 30 min, followed by primary mouse anti-ERα antibody (dilution 1:100, C-311, Santa Cruz Biotechnology Co., Ltd.) and mouse anti-ERβ (dilution 1:100, B1, Santa Cruz Biotechnology Co., Ltd.), respectively, overnight at 4 °C. Sections incubated in phosphate-buffered saline (PBS) without antibody served as negative controls. Then objective tissue was covered with secondary antibody (m-IgGκ BP-HRP, dilution 1/50, Santa Cruz Biotechnology Co., Ltd.) to appropriately respond to primary antibody in species, and incubated at room temperature for 50 min. The sections were stained with 3,3'diaminobenzidine (DAB) (Sigma). The nucleus was stained, and the section was mounted with resin mounting medium. The Image-Pro Plus 6.0 image analysis system was used for quantitative analysis.
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