Ecl hrp chemiluminescent substrate reagent kit

Cultured or purified cells were collected and lysed. The protein concentration was measured with a bicinchoninic acid protein assay kit (Beyotime). The protein sample was separated in 10% SDS-denatured polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membranes were blocked with 5% skim milk in TBST at room temperature for 2 h. The targeted molecules were probed using specifc primary Abs and HRP-conjugated secondary Abs and were detected with an ECL HRP chemiluminescent substrate reagent kit (Invitrogen, Carlsbad, CA).

Paraffin sections were dewaxed by xylene, followed by antigen repair in 0.01 M citrate buffer. After wash, the sections were blocked with 5% BSA (Amresco, Pittsburgh, PA, USA). The slides were then incubated overnight at 4 °C with the purified anti-EmFBA1 antibodies diluted at 1:100. After wash, Alexa Fluor 594 goat anti-rabbit antibodies diluted at 1:10,000 (Merck) were added and incubated at room temperature for 1 h, followed by overnight incubation with DAPI (Merck). The slides were observed under fluorescence microscope (Leica, Berlin, Germany).
Western blotting was conducted as previously described [20 (link)]. Briefly, 10 μg of the crude tapeworm proteins (E. multilocularis, E. granulosus, Taenia hydatigena and Taenia asiatica) stored in our laboratory was resolved using 10% SDS-PAGE gel, and then transferred to PVDF membrane (Millipore, Burlington, MA, USA). The membrane was sequentially incubated with 1:1000 diluted purified anti-EmFBA1 or 1:10,000 diluted anti-acitn (Abcom, London, UK) and then 1:10,000 diluted goat anti-rabbit IgG HRP linked (Sera care, Gaithersburg, MD, USA). After wash, the membrane was dealt with ECL HRP Chemiluminescent Substrate Reagent kit (Invitrogen, Carlsbad, CA, USA) and then exposed to X-ray film (Carestream, Rochester, NY, USA) for visualization. In this experiment, healthy rabbit serum was used as control.

These experiments were performed following previously described procedures (7 (link)). Briefly, RNA was extracted with an RNase Mini Kit and cDNA was synthesized with a SuperScript III Reverse Transcriptase (QIAGEN, Valencia, CA, USA). qRT-PCR was performed with 2.5 μL of cDNA, 12.5 μL of SYBR Master Mixture (Applied Biosystems, Foster City, CA, USA), and target gene–specific primers (Table 1). Amplification of β-actin was used as an internal control. For western blotting, the cultured or purified cells were collected and lysed. The protein concentration was measured with a bicinchoninic acid protein assay kit (Beyotime). The protein sample was separated in 10% SDS-denatured polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The polyvinylidene difluoride membranes were blocked with 5% skim milk in TBST at room temperature for 2 h. The targeted molecules were probed using specific primary antibodies and HRP-conjugated secondary antibodies and detected with an ECL HRP chemiluminescent substrate reagent kit (Invitrogen, Carlsbad, CA).