The splenic samples were homogenized in RIPA Lysis Buffer (Biosharp, BL504A) with protease inhibitor. The protein concentration was calculated using a BCA Protein Assay kit (Tiangen Biotech), and total proteins were separated by electrophoresis on 12% SDS-PAGE gels. Total proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA), and then the membranes were blocked with 5% skimmed milk powder. The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively. After washing, the membranes were incubated with goat anti-mouse IgG-HRP (Biosharp, BL001A) or rabbit anti-goat IgG-HRP (Biosharp, BL004A) with 1:10 000 dilution, and the signals were detected using an ECL western blotting detection reagent (Tiangen Biotech). The immunospecific bands were quantified using Quantity One V452 (Bio-Rad Laboratories, Hercules, CA, USA) with GAPDH as an internal control protein. GAPDH was detected using an anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., sc-20357, 1:1000).
Quantity one v452
Manufactured by Bio-Rad
Sourced in United States
The Quantity One V452 is a software application for image analysis and quantification. It provides tools for capturing, analyzing, and managing digital images from a variety of laboratory equipment, including gel documentation systems, Western blot imagers, and other imaging devices.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh antibody, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab230428, by Abcam (1 mentions)
Mouse anti mx1 monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Tiangen Biotech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Goat anti mouse igg hrp, by Biosharp (1 mentions)
Pro light hrp chemiluminescence detection reagent, by Tiangen Biotech (1 mentions)
Sc 20357, by Santa Cruz Biotechnology (1 mentions)
0.22 μm pvdf membrane, by Merck Group (1 mentions)
2720 thermocycler, by Thermo Fisher Scientific (1 mentions)
Puretaq ready to gotm pcr beads, by GE Healthcare (1 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Red safe 1x, by iNtRON Biotechnology (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Ready to go pcr beads, by GE Healthcare (1 mentions)
Abi prism 310, by Thermo Fisher Scientific (1 mentions)
6 protocols using quantity one v452
1
Splenic Protein Expression Analysis
2
Analysis of NF-κB Protein Expression
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The total proteins from thymic samples were extracted as described previously19 (link). The protein samples (10 μg/lane) were separated on 12% SDS-PAGE gels, and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Blot analysis was performed with a mouse anti-NF-κB1 monoclonal antibody (Santa Cruz Biotechnology, Inc., sc-8414, 1:1000), a mouse anti-NF-κB2 monoclonal antibody (Santa Cruz Biotechnology, sc-7386, 1:1000), a mouse anti-RelA monoclonal antibody (Santa Cruz Biotechnology, sc-8008, 1:1000), a mouse anti-RelB monoclonal antibody (Santa Cruz Biotechnology, sc-166416, 1:1000), and a mouse anti-c-Rel monoclonal antibody (Santa Cruz Biotechnology, sc-6955, 1:1000) at 4 °C overnight, respectively. Then, the membranes were washed and incubated with goat anti-mouse IgG-HRP (horseradish peroxidase) secondary antibody (Biosharp, BL001A, 1:2000) for 1 h at room temperature. Chemiluminescent substrate was used according to the manufacturer’s instructions (Tiangen Biotech) to detect immunoreactive protein. An anti-GAPDH antibody (Santa Cruz Biotechnology, sc-20357, 1:1000) was applied as an internal control protein. The blots were exposed to X-ray film, and densitometry of autoradiograms was performed using Quantity One V452 (Bio-Rad Laboratories). Relative expression levels of target proteins were normalized using the GAPDH.
3
Cytokine Expression Analysis by Western Blot
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The total proteins were extracted, and the protein concentration was measured as described previously [8 (link)]. Total proteins (10 μg/lane) were separated by SDS-PAGE followed by transferring onto 0.22 μm PVDF membranes (Millipore, Bedford, MA, USA). Blot analysis was performed with an anti-IFN-γ antibody (Abcam, San Francisco, CA, USA, ab27919), an anti-TNF-β antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, SC-28345), an anti-IL-2 antibody (Abcam, ab193807), an anti-IL-4 antibody (Bio-Techne, MAB2469), an anti-IL-5 antibody (Santa Cruz Biotechnology, SC-8433), an anti-IL-6 antibody (Abcam, ab193853), and an anti-IL-10 antibody (Santa Cruz Biotechnology, SC-32815) at a dilution of 1:1000 to analyze expression of Th1 and Th2 cytokines respectively. Target proteins were detected by a Pro-light HRP chemiluminescence detection reagent (Tiangen Biotech, Beijing, China). An anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, sc-20357, 1:1000) was used for normalization of sample loading. The intensity of blots were semi-quantified by Quantity One V452 (Bio-Rad Laboratories), and the values were calculated using GAPDH as an internal reference.
4
Ovine Splenic Protein Isolation and Western Blot Analysis
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The proteins were isolated from ovine splenic samples as described previously [13 (link)]. Samples were isolated by SDS-polyacrylamide gel electrophoresis and transferred electrophoretically on PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with skim milk, and incubated with the antibodies (Table S2 ) at a dilution of 1:1000. Then, the membranes were incubated with an HRP-conjugated anti-mouse secondary antibody (Biosharp, Hefei, China BL001A, 1:2000). An HRP chemiluminescence kit was applied for detecting blots. The blot intensity was analyzed by Quantity One V452 (Bio-Rad Laboratories)., Hercules, CA, USA). The relative intensity of the blots was calculated and normalized with a reference protein (GAPDH) using an anti-GAPDH antibody (Table S2 , 1:1000).
5
PCR Amplification and Sequencing of Cytochrome b
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PCR reactions were performed in a final volume of 25 μL using PureTaq TM Ready-to-Go TM PCR beads (GE Healthcare UK Ltd) with the addition of molecular-grade water, primers (final concentration 0.2 µM) and 50 ng of DNA template. The thermal cycling parameters were as follows: 95°C for 5 min, followed by 25 cycles of 95°C for 20 s, 69°C for 30 s, 72°C for 30 s and a terminal extension step of 72°C for 5 min. Negative controls (molecular-grade water) were included in each set of reactions. These PCR reactions were carried out in an Applied Biosystems 2720 Thermocycler.
The amplification products were tested in a 2% agarose gel (Pronadisa), containing RedSafe™ 1X (iNtRON Biotechnology) in 0.5X TBE buffer (Sigma). DNA fragments were visualized using the Gel Doc XR System and the software Quantity One® v 4.5.2 (Bio-Rad).
Sequencing was used to verify the correct species assignation of the specimens used for the LFD method set-up. L14735 and H1549D (Kocher et al, 1989) were used for the amplification and subsequent sequencing on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The resulting Cytochrome b nucleotide sequences were analyzed using Megablast with those present in the NCBI database.
The amplification products were tested in a 2% agarose gel (Pronadisa), containing RedSafe™ 1X (iNtRON Biotechnology) in 0.5X TBE buffer (Sigma). DNA fragments were visualized using the Gel Doc XR System and the software Quantity One® v 4.5.2 (Bio-Rad).
Sequencing was used to verify the correct species assignation of the specimens used for the LFD method set-up. L14735 and H1549D (Kocher et al, 1989) were used for the amplification and subsequent sequencing on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The resulting Cytochrome b nucleotide sequences were analyzed using Megablast with those present in the NCBI database.
6
Validating RT-PCR Analysis Using Cytb Sequencing
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In order to check the reliability of the RT-PCR analysis and test the functionality of the DNA extracted, a 464 bp fragment of cytb gene from all reference and commercial samples was amplified and sequenced using the primers H15149: 5'-GCICCTCARAATGAYATTTGTCCTCA-3' and L14735: 5'-AAAAACCACCGTTGTTATTCAACTA-3' (Burgener, 1997) .
PCR reactions were carried out in a total volume of 25 µL with 100 ng of DNA template, using PCR Ready-to-Go beads (GE Healthcare) with a final concentrations of 640 nM for forward and reverse primers on a Applied Biosystems 2720 thermocylcer.
The thermal cycling protocol used was: 94ºC for 5 min; followed by 35 cycles of 94ºC for 40 s, 55ºC for 80 s, 72ºC for 80 s; and a final extension step of 72ºC for 7 minutes.
PCR amplicons were visualized on a 2% agarose gel, using the Gel Documentation System Gel Doc XR System and the software Quantity One ® v 4.5.2 (Bio-Rad).
These PCR products were sequenced on an ABI PRISM 310 genetic analyser (Applied Biosystems) using the primers described above and reactions the BigDye Terminator 1.1 (Applied Biosystems), following the supplier's instructions and sequencing. The nucleotidic sequences were analysed using BioEdit (Hall, 1999) and MEGA (Kumar, Dudley, Nei & Tamura, 2008) software and the results were authenticated with a BLAST analysis (Alschul et al., 1997) .
PCR reactions were carried out in a total volume of 25 µL with 100 ng of DNA template, using PCR Ready-to-Go beads (GE Healthcare) with a final concentrations of 640 nM for forward and reverse primers on a Applied Biosystems 2720 thermocylcer.
The thermal cycling protocol used was: 94ºC for 5 min; followed by 35 cycles of 94ºC for 40 s, 55ºC for 80 s, 72ºC for 80 s; and a final extension step of 72ºC for 7 minutes.
PCR amplicons were visualized on a 2% agarose gel, using the Gel Documentation System Gel Doc XR System and the software Quantity One ® v 4.5.2 (Bio-Rad).
These PCR products were sequenced on an ABI PRISM 310 genetic analyser (Applied Biosystems) using the primers described above and reactions the BigDye Terminator 1.1 (Applied Biosystems), following the supplier's instructions and sequencing. The nucleotidic sequences were analysed using BioEdit (Hall, 1999) and MEGA (Kumar, Dudley, Nei & Tamura, 2008) software and the results were authenticated with a BLAST analysis (Alschul et al., 1997) .
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