ASFV-infected CAS-01 cells were subjected to ICC assay. In order to fix the cells, 80% Acetone was added on the cells after removing the media from the plate. The cells were allowed to dehydrate and fix for 10 min after adding the acetone. Afterward, acetone was removed and 10 min of drying were undertaken at room temperature. Next, cells were washed once with PBS, and rabbit Anti-ASFV p30 polyclonal antibody (Creative-diagnostics™, CABT-RM033) was added as the primary antibody at a 1:500 dilutions and incubated at 37 °C for 1 h. Following 4 times washing with PBS, polyclonal goat anti-rabbit immunoglobulin (Enzo™, ADI-SAB-301-J) was used as a secondary antibody at a 1:1000 dilution and incubated at 37 °C for 1 h. Finally, cells were washed 4 times with PBS and detected using alkaline phosphatase substrate (ImmPACT® Vector® Red Substrate, Alkaline Phosphatase, Vector, SK-5100). The substrate was washed aware with distilled water to avoid over-staining.
Adi sab 301 j
Manufactured by Enzo Life Sciences
The ADI-SAB-301-J is a laboratory instrument manufactured by Enzo Life Sciences. It is designed for performing analytical tasks within a research or testing environment. The core function of this product is to support various experimental procedures, but a detailed description of its specific capabilities or intended applications is not available.
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2 protocols using adi sab 301 j
1
ICC Assay for ASFV Detection
2
FAP-α Protein Expression Analysis
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Total protein was extracted from rAd-FAP-α DCs, rAd-c DCs, LLC cells or CAFs using RIPA buffer [150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA, 10 mM NaF, 1 mM sodium vanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol] (eBioscience; Thermo Fisher Scientific, Inc.) on ice for 30 min. Protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Proteins (20 µg) were separated by 6% SDS-PAGE and transferred onto Hybond-polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in PBS at room temperature for 1 h and subsequently incubated with primary antibodies against FAP-α (1:1,000; cat. no. NB110-85534; Novus Biologicals, Ltd.) and GAPDH (1:1,000; cat. no. NB100-56875; Novus Biologicals, Ltd.) for 2 h at room temperature. Membranes were washed three times in TBST (5 min/wash) and further incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody (1:2,500; cat. no. ADI-SAB-301-J; Enzo Life Sciences Inc.) for 1 h at room temperature. Protein bands were visualized using the ECL western blot analysis system (GE Healthcare).
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