To determine viral titers, 3.2 × 105 VeroE6 or VeroE6-TMPRSS2 were seeded in a 12 well-plate the day before plaque assay was performed. Briefly, ten-fold serial dilutions were performed in infection media for SARS-CoV-2 and inoculated onto confluent VeroE6 or VeroE6-TMPRSS2 cell monolayer. After one-h adsorption, supernatants were removed, and cells monolayers were overlaid with minimum essential media (MEM) containing 2% FBS and purified agar (OXOID) at a final concentration of 0.7%. Cells were then incubated 3 days at 37°C. Cells were fixed overnight with 10% formaldehyde for inactivation of potential SARS-CoV-2 virus. Overlay was removed and cells were washed once with PBS. Plaques were visualized by immunostaining. Briefly, cells were blocked in 5% milk diluted in TBST. After 1 h, anti-mouse SARS-CoV-2- NP antibody (1C7C7, kindly provided by Dr. Moran) was added at a dilution of 1:1000 in 1% milk-TBST and incubated for 1 h at RT. Then, cells were washed two times in PBS and stained with goat anti-mouse secondary IgG-HRP antibody (abcam, 6823) at a dilution of 1:5000 in 1% milk-TBST and incubated for 1 h at RT. Finally, cells were washed three times and the plaques were developed with TrueBlue substrate (KPL-Seracare, 5510-0030). Viral titers were calculated considering number of cells per well and expressed as plaque forming units (PFU)/mL.
Goat anti mouse igg hrp secondary antibody
Manufactured by Abcam
Sourced in United States
Goat anti-mouse IgG-HRP secondary antibody is a reagent used in immunoassays and other laboratory techniques. It is a polyclonal antibody produced in goats that binds to mouse immunoglobulin G (IgG) antibodies. The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that can be used to detect and quantify target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Anti cd19 anti cd3 bispecific antibody, by BPS Biosciences (1 mentions)
Streptavidin coated plate, by Thermo Fisher Scientific (1 mentions)
7 protocols using goat anti mouse igg hrp secondary antibody
1
SARS-CoV-2 Plaque Assay Protocol
2
SARS-CoV-2 Plaque Assay Protocol
3
ECE2 Immunohistochemistry Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For IHC staining, sections were deparaffinized in xylene and rehydrated in alcohol, then endogenous peroxidase was inactivated by treatment with 3% H2O2 for 5 min. Then, antigen retrieval was performed in a pressure cooker with sodium citrate buffer (pH 6.0). After blocking with 5% goat serum for 30 min at room temperature, sections were incubated with rabbit ECE2 monoclonal antibody (1:200, Abcam, USA) overnight in a 4°C refrigerator. Sections were coupled with goat anti-mouse IgG-HRP secondary antibody (1:400, Abcam, USA) for 1 hour at room temperature, then each incubated section was stained with DAB reagent and finally counterstained with hematoxylin (12 (link)).
4
GLUT1 Expression in Esophageal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Clinical samples were obtained from 50 patients with ESCA who were surgically treated at Taihe Hospital Affiliated of Hubei University of Medicine from February 2016 to September 2017. The content of GLUT1 was detected by IHC according to the method previously described (9 (link)). The ESCA tissue and the paracarcinoma tissues were prepared into 3 μm paraffin sections and incubated with mouse monoclonal antibodies of GLUT1 (1:200, Abcam, USA) at 4°C overnight in a refrigerator. The sections were coupled with the goat anti-mouse IgG-HRP secondary antibody (1:2000, Abcam, USA) at room temperature for 1.5 h, then each incubated section was stained with DAB reagent, and finally counterstained with hematoxylin.
5
Immunofluorescence Analysis of Exosome-Induced Macrophage Polarization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PMA-induced THP-1 cells and RAW 264.7 cells treated with corresponding NC or ferroptosis-induced MDA-MB-231 cell and 4T1 cell derived-exosomes were seeded on cell slides and incubated overnight. Next, the cells were fixed in 4% paraformaldehyde at room temperature for 30min, permeabilized with 0.5% Triton X-100, and blocked with 1% bovine serum albumin (GIBCO, Waltham, MA, USA). The cells were incubated overnight at 4 °C with primary antibody against CD86 (1:100; Invitrogen) and Arg-1 (1:500; Proteintech). After washing with PBS, the cells were incubated with goat anti-mouse IgG-HRP secondary antibody (1:500, Abcam) for 1 h at room temperature. DAPI (Abcam) was used to stain cell nucleus. After washing 3 time with PBS, the sections were performed for photograph under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
6
Measuring mAb-CD47 Receptor Binding Affinity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The binding affinity of the mAb-CD47 receptor was tested by measuring the dissociation constant (KD) following the reported procedure [60 (link)]. Briefly, the 96-well plate was coated with 200 ng of CD47 receptor in 100 µL of coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) and incubated at 4 °C overnight. The plate was washed three times with PBS containing 0.05% Tween 20 and blocked with 2% BSA (bovine serum albumin). Then, 100 µL of three equilibrated reactions of anti-CD47 mAb and receptor with nanomolar ratios of 2.22:4.44, 3.33:3.33, and 4.44:2.22 and anti-CD47 mAb with concentrations of 0, 1, 5, 10, 20, 40, 80, 160, 320, and 640 nM (for standard curve generation) were added into each well. After incubating at 37 °C for 1 h, 100 µL of HRP goat anti-mouse IgG secondary antibody (Abcam, Cambridge, MA, USA) at a concentration of 50 ng/mL was added to each well and incubated at 37 °C for 1 h and followed with TMB substrate for color development. The reaction was stopped by adding 100 µL of 1 M sulfuric acid. The absorbance was recorded with a BioTek plate reader at a wavelength of 450 nm.
7
Quantifying CD19 sBite in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure and detect the presence of the CD19 sBite in culture media, an ELISA and western blot was performed. First, γδ T cells were electroporated and cultured overnight (∼16 h) and the conditioned media was collected. To perform the ELISA, streptavidin-coated plates (Fisher) were coated with biotinylated human CD3ε and CD3δ heterodimer protein with His/Avitag (Acro Biosystems). Conditioned media samples were then added to the plate, with an anti-CD19-anti-CD3 bispecific antibody (BPS Biosciences) used as a standard. Next, a CD19Fc fusion protein (R&D Systems) was added to the plates, followed by horseradish peroxidase (HRP) anti-human Fc (Jackson Labs). Finally, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution (Fisher) was added to the plate and the absorbance was measured at 450 nm.
To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions. Next, separation by SDS-PAGE and transfer to a nitrocellulose membrane was performed. The blocked membrane was incubated with an anti-His antibody (R&D Research), followed by an HRP goat anti-mouse IgG secondary antibody (Abcam).
To perform the western blot, γδ T cell conditioned media and anti-CD19-anti-CD3 bispecific antibody standards (BPS Biosciences) were prepared under reducing conditions. Next, separation by SDS-PAGE and transfer to a nitrocellulose membrane was performed. The blocked membrane was incubated with an anti-His antibody (R&D Research), followed by an HRP goat anti-mouse IgG secondary antibody (Abcam).
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!