MERS-CoV–specific antibody titers were measured by ELISA. First, 96-well plates were coated with MERS-CoV S1 or MERS-CoV RBD proteins at 1 μg/ml in PBS (pH 7.4) and incubated overnight at 4°C. Wells were then washed three times with PBS, blocked with 10% normal goat serum in PBS, and incubated at 37°C for 30 min. Dromedary camel sera or VHHs were serially diluted in PBS, 100 μl was added per well, and plates were incubated at 37°C for 1 hour. Next, plates were washed three times in PBS containing 0.05% Tween 20 (PBST), after which they were incubated with biotin-conjugated goat anti-llama antibodies (1:2000, Abcore) or mouse anti-histidine antibodies (1:2000, Thermo Fisher Scientific) at 37°C for 1 hour. After three washes with PBST, plates were incubated with streptavidin horseradish peroxidase (HRP; 1:10,000, Dako) or goat anti-mouse HRP (1:2000, Dako) at 37°C for 1 hour. After this incubation, plates were washed three times in PBST and incubated at room temperature for 10 min in the presence of 3,3′,5,5′-tetramethylbenzidine substrate (eBioscience). Reactions were stopped with 2N H2SO4 (Sigma). The absorbance of each sample was read at 450 nm with an ELISA reader (Tecan Infinite F200).
Streptavidin horseradish peroxidase hrp
Manufactured by Agilent Technologies
Sourced in Denmark
Streptavidin-horseradish peroxidase (HRP) is a conjugate of the protein streptavidin and the enzyme horseradish peroxidase. It is a commonly used reagent in various bioanalytical and immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Hybridoma alpha ca cells, by American Type Culture Collection (2 mentions)
Biocoat coverslips, by Corning (2 mentions)
Dmr microscope, by Leica (2 mentions)
3 3 5 5 tetramethylbenzidine substrate, by Thermo Fisher Scientific (1 mentions)
2 n h2so4, by Merck Group (1 mentions)
Goat anti mouse hrp, by Agilent Technologies (1 mentions)
Infinite f200, by Tecan (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Nis elements software, by Nikon (1 mentions)
Paraformaldehyde solution, by Fujifilm (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Agilent Technologies (1 mentions)
Abiorevo bz 9000 microscope, by Keyence (1 mentions)
5 protocols using streptavidin horseradish peroxidase hrp
1
MERS-CoV Antibody Titer Quantification
2
Quantification of Dystrophin-Positive Muscle Fibers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Muscles samples were snap-frozen in isopentane cooled in liquid nitrogen and stored at <−70 °C until processing. Dystrophin-positive fibres were numbered in a blinded manner on serial transverse sections after immunohistochemical revelation of dystrophin using a mouse monoclonal anti-dystrophin antibody (Novocastra NCL-DYS-B, clone 34C5, 1:50, Leica). Goat anti-mouse biotinylated IgG (1:300, Dako) was used as secondary antibody, diluted in 5% dog serum in phosphate-buffered saline (PBS). The sections were then incubated with streptavidin/horseradish peroxidase (HRP) (1:300, Dako) and then revealed using 3,3′-diaminobenzidine (DAB, Dako). Although dystrophin-positive fibres in positive samples were scattered all along the muscle samples as individual or more often as clustered fibres, a minimum of 3 microscopic fields at intermediate magnification were randomly chosen to finally observe a minimum of 250 fibres (intraobserver variation coefficient was below 5%). All measurements were performed using Nikon’s NIS-Elements software (Nikon). Statistical analyses were performed using the nonparametric Mann–Whitney test. A difference was considered to be significant at *P<0.05, **P<0.01 or ***P<0.001.
3
Quantifying MLV Titers by Coculture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the quantification of MLV titers by coculture, round, 12-mm-diameter BioCoat coverslips (Corning; Fisher Scientific, Merelbeke, Belgium) were first added to a 24-well plate (Nunc; Thermo Scientific, Asse, Belgium). Next, approximately 1.5 × 105 NIH 3T3 cells were seeded into each well. After 24 h, NIH 3T3 cells were cocultured with 104 cells from the spleen or thymus for 24 h. Next, NIH 3T3 cells were rinsed with PBS and fixed with 4% paraformaldehyde. For staining, a primary antibody against the MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). The primary antibody was incubated for 1 h. Goat anti-mouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody, and samples were incubated for 20 min, followed by an incubation step with streptavidin-horseradish peroxidase (HRP) (Dako Denmark) for 30 min. Next, 3,3’-diaminobenzidine (DAB) staining was done. Coverslips were mounted using Mowiol 4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (×40 magnification).
4
Quantifying MLV Titers by Coculture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the quantification of MLV titers by coculture, round, 12-mm-diameter BioCoat coverslips (Corning; Fisher Scientific, Merelbeke, Belgium) were first added to a 24-well plate (Nunc; Thermo Scientific, Asse, Belgium). Next, approximately 1.5 × 105 NIH 3T3 cells were seeded into each well. After 24 h, NIH 3T3 cells were cocultured with 104 cells from the spleen or thymus for 24 h. Next, NIH 3T3 cells were rinsed with PBS and fixed with 4% paraformaldehyde. For staining, a primary antibody against the MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). The primary antibody was incubated for 1 h. Goat anti-mouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody, and samples were incubated for 20 min, followed by an incubation step with streptavidin-horseradish peroxidase (HRP) (Dako Denmark) for 30 min. Next, 3,3’-diaminobenzidine (DAB) staining was done. Coverslips were mounted using Mowiol 4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (×40 magnification).
5
Immunohistological Analysis of H-D Antigen
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A homozygous GalT/CMAH double KO pig (M129-2), a homozygous
GalT KO pig (M71-5) and an age-matched wild-type (WT) pig were sacrificed 2 days after
birth. Harvested samples were used for immunohistological analysis of the H-D antigen. The dissected organs
were fixed in a 4% paraformaldehyde solution (Wako Pure Chemical Industries, Osaka, Japan), embedded in
paraffin, sectioned and stained with hematoxylin using standard methods.
The fixed sections were also incubated with blocking solution (2% BSA/D-PBS) for 1 h and then treated with a
chicken anti-H-D antibody (a generous gift from Prof N Wakamiya, Asahikawa Medical University) for 1 h. After
removal of the excess antibody, the sections were incubated with biotin-conjugated goat anti-chicken IgY
(Abcam, Cambridge, United Kingdom) for 30 min. For detection of the α-Gal antigen, the sections were incubated
for 1 h with biotin-conjugated Griffonia simplicifolia I (GS-IB4) lectin (Life Technologies).
Each section was also incubated with Streptavidin-Horseradish Peroxidase (HRP) (DAKO, Tokyo, Japan) for 15 min
and 3,3′-Diaminobenzidine tetrahydrochloride (DAB) (DAKO) for 5 min. The slides were visualized using a
Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
GalT KO pig (M71-5) and an age-matched wild-type (WT) pig were sacrificed 2 days after
birth. Harvested samples were used for immunohistological analysis of the H-D antigen. The dissected organs
were fixed in a 4% paraformaldehyde solution (Wako Pure Chemical Industries, Osaka, Japan), embedded in
paraffin, sectioned and stained with hematoxylin using standard methods.
The fixed sections were also incubated with blocking solution (2% BSA/D-PBS) for 1 h and then treated with a
chicken anti-H-D antibody (a generous gift from Prof N Wakamiya, Asahikawa Medical University) for 1 h. After
removal of the excess antibody, the sections were incubated with biotin-conjugated goat anti-chicken IgY
(Abcam, Cambridge, United Kingdom) for 30 min. For detection of the α-Gal antigen, the sections were incubated
for 1 h with biotin-conjugated Griffonia simplicifolia I (GS-IB4) lectin (Life Technologies).
Each section was also incubated with Streptavidin-Horseradish Peroxidase (HRP) (DAKO, Tokyo, Japan) for 15 min
and 3,3′-Diaminobenzidine tetrahydrochloride (DAB) (DAKO) for 5 min. The slides were visualized using a
Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!