Immunofocus assays on Vero cells were used to quantify infectious DTV as previously described (57 (link)). Briefly, Vero cells were seeded 1 day prior to infection in 12- or 24-well plates at 1 × 105 or 5 × 104 cells per well, respectively. Serum or homogenized tissue was serially diluted 1:10 in DMEM. Medium was removed from the Vero cells and replaced with diluted sample. Plates were incubated at 37°C with gentle rocking for 1 h before inoculum was aspirated. Cells were washed once with 1× phosphate-buffered saline (PBS), and an overlay of 0.8% methylcellulose in DMEM was added. After 6 days incubation at 37°C, the cells were fixed with 100% methanol. Cells were incubated with Powassan hyperimmune ascites fluid (ATCC) diluted 1:1,000 in Opti-MEM and anti-mouse horseradish peroxidase-conjugated secondary antibody (Dako) diluted 1:1,000 in Opti-MEM for 1 h each; then, plaques were developed using diaminobenzidine (Sigma) and 0.0135% H2O2 (Sigma). Foci were counted and used to calculate viral titer (log10 FFU) per milliliter of serum or per gram of tissue.
Horseradish peroxidase conjugated anti mouse secondary antibody
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The Horseradish peroxidase-conjugated anti-mouse secondary antibody is a laboratory reagent used in immunoassays and western blotting. It is designed to detect and bind to primary antibodies raised against mouse antigens, allowing for visualization and quantification of target proteins.
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Lab products found in correlation
Liquid 3 3 diaminobenzidine dab substrate, by Agilent Technologies (2 mentions)
Evans blue, by Merck Group (2 mentions)
Acetylated tubulin, by Merck Group (1 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Tyrosinated tubulin, by Merck Group (1 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (1 mentions)
Luminata forte western horseradish peroxidase hrp substrate, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by AG Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Sigmafast opd peroxidase substrate, by Merck Group (1 mentions)
Hybond p polyvinylidene difluoride pvdf membrane, by Cytiva (1 mentions)
Ecl plus, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Axiocam erc5s digital camera, by Zeiss (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Mayer s hematoxylin solution, by Agilent Technologies (1 mentions)
Ab236882, by Abcam (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Merck Group (1 mentions)
13 protocols using horseradish peroxidase conjugated anti mouse secondary antibody
1
Quantifying Infectious DTV in Vero Cells
2
Axon Protein Extraction and Quantification
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Plating media was removed from microfluidic chambers and culture was rinsed with HBSS. Axons were harvested with RIPA buffer with protease (CompleteTM Mini Protease Inhibitor Cocktail tablets, Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail, A.G. Scientific), with protein pooled from 2 to 4 chambers per treatment group. Denatured protein samples (20 μg) were electrophoresed into Bolt®; 4–12% Bis-Tris gels (Invitrogen), transferred to PVDF membrane (Bio-Rad) and incubated overnight with primary antibodies acetylated tubulin (1:1,000 mouse, Sigma), tyrosinated tubulin (1:1,000 rabbit, Millipore), MAP1B (1:1,000 mouse, AbCam). The corresponding anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibody (1:7,000, DAKO) was used as previously described (King et al., 2018 (link)). GAPDH (1:5,000 mouse, Millipore) was used as a loading control. Bands were visualized with enhanced chemiluminescence (ECL) solution-Luminata Forte Western horseradish peroxidase (HRP) substrate (Millipore) and images acquired with a Chemi-Smart 5000 Imaging System (Vilber Lourmat) equipped with Chemi-Capt 5000 software. Band intensity was measured as the integrated intensity using Fiji software. After standardizing to GAPDH, each value was calculated as a percentage of control samples (See Supplementary Figure 1 ).
3
Exosome Immunoassay Protocol
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Exosomes (40 µg) were plated in wells of a 96-well plate. In addition, 50 µL buffer was plated as a control. Then, 100 µL bicarbonate buffer with a pH of 9.5 was added to each well, and plates were incubated at 4 °C overnight. The next day, plates were washed with 1 × PBS with 0.05% Tween 20 three times. After washing, plates were blocked with 5% skim milk in PBS with 0.09% Tween 20 at 4 °C for 1 h. After blocking, primary antibodies were added to each well, and plates were incubated at room temperature (RT) for 2 h or at 4 °C overnight. After incubation, the plates were rewashed as described above, and horseradish peroxidase-conjugated secondary antibody (anti-mouse, 1:1000 or anti-rabbit, 1:1000; Dako) was added to each well for incubation at RT for 2 h or at 4 °C overnight. After incubation, the ELISAs were developed with SigmaFast™ OPD peroxidase substrate (Sigma Aldrich) in the dark at RT for 30 min. Intensities were determined at 405 nm using a Genemate UniRead 800 microplate reader.
4
Immunoblotting Analysis of Lung Proteins
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Homogenized lungs were lysed on ice in 500 μl lysis buffer [20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2 mM EDTA (pH 7.4), 1% Triton X-100, 10% glycerol, 1 mM sodium vanadate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 25 mM glycerophosphate and 10 μg/ml leupeptin]. Lysates were cleared by centrifugation and the protein concentration was determined. SDS-PAGE gel electrophoresis of the cell lysates were performed as described [29 (link)]. After electrophoresis, proteins were transferred to pretreated Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were incubated for 1h at room temperature in blocking buffer (5 % non-fat dry milk in PBS containing 0.1% Tween) and subsequently for 12 h at 4°C in blocking buffer with primary antibodies raised against β-actin (1/5000, Sigma) or FGF2 (clone FB-8, 1/5000, Sigma). After washing, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (anti-mouse: 1/2000 or anti-rabbit: 1/4000, Dako) in blocking buffer for 25 min at room temperature. Next, the membranes were washed extensively. Immunoreactive proteins were detected by chemiluminescence (ECLplus, Bio-Rad). Samples were collected from 3 independent experiments.
5
Quantifying Cytokeratin Expression in 3D Tissues
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Paraffin‐embedded 3‐D tissues were sectioned at a thickness of 6 µm; the sections were stained with hematoxylin and eosin. For immunohistochemistry, sections were deparaffinized, and incubated for 5 min at 20°C with proteinase K (Dako, Glostrup, Denmark) diluted 1:1000 in phosphate‐buffered saline [PBS (−)] followed by 3% hydrogen peroxide. After washing, sections were incubated in 1% bovine serum albumin for at least 20 min at room temperature. A mouse monoclonal antibody against human cytokeratin (CK) (Dako) diluted 1:500 was added overnight at 4°C, followed by incubation for 30 min at room temperature with horseradish peroxidase‐conjugated antimouse secondary antibody (Dako). After washing with PBS (−), staining with diaminobenzidine (Dako), and nuclear staining with Mayer's hematoxylin for 30 s, sections were washed and imaged under a light microscope (400× magnification) with an AxioCamERc5s digital camera (Carl Zeiss, Oberkochen, Germany). Three randomly selected views were imaged for each of the three specimens. The average of number of CK‐positive cells that reached the membrane at the bottom of the 3‐D tissue was determined.
6
STAT1 Immunohistochemistry in Skin Biopsies
7
Histological Analysis of Engineered Cartilage
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The implants harvested at investigated time points and normal cartilage tissue of pig ears were collected for further histological staining as described previously (Liu et al., 2008 (link)). Briefly, samples were fixed in 4% paraformaldehyde, paraffin-embedded, and sliced into 5-μm-thick sections. HE staining was performed to analyze the structure of implants. Toluidine blue and Safranin O staining were used to evaluate GAG deposition in the engineered cartilage tissues. Collagen II expression of pig chondrocytes was specifically detected by a mouse anti-human collagen II antibody (1:200; Abcam, Cambridge, MA, United States) and horseradish peroxidase-conjugated anti-mouse secondary antibody (1:50; Dako, Denmark). The sections were developed using diaminobenzidine tetrahydrochloride (DAB, Dako, Denmark), and images were acquired by microscope (Olympus BX51, Japan) and at least six representative fields from each sample were examined.
8
Immunohistochemical Analysis of FAM83D in ESCC
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ESCC and para-carcinoma tissue samples were collected from sixty-nine patients with ESCC who came to the Department of Thoracic Surgery, the Fourth Hospital of Hebei Medical University (Hebei, China) from January 2010 to December 2012. All the patients signed informed consent forms, and all protocols in this study were approved by the ethics committee of the Fourth Hospital of Hebei Medical University. A standard immunohistochemical analysis protocol was applied using the streptavidin-peroxidase method as previously described.11 (link) Briefly, after antigen retrieval, the slides were incubated with an anti-FAM83D monoclonal antibody (1:200; ab236882, Abcam, USA) followed by incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:200; Dako, Denmark). Subsequently, antibody binding was visualized after diaminobenzidine coloring reaction and hematoxylin counterstaining. PBS acted as a negative control instead of the primary antibody. The immunostained results were independently evaluated by two clinical pathologists according to the scoring standard of previous studies.12 (link)
9
Liver Immunohistochemistry for AREG Expression
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Mouse livers were excised, fixed in 4% formalin, and embedded in paraffin. Sectioned tissues were stained with hematoxylin and eosin. Immunohistochemical staining was performed by incubating the tissue sections with a primary antibody against AREG (dilution 1 : 500) in a moisturized chamber at 4°C overnight. The membranes were then incubated with a horseradish peroxidase-conjugated anti-mouse secondary antibody (Dako, Glostrup, Denmark). Stained cells were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
10
Western Blot Analysis of COX-IV
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Equal amount of supernatant from the pellet of cell culture medium were loaded onto 4–12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad laboratories, Hercules, CA, USA). The membranes were incubated with blocking buffer containing 0.1% Tween-20 and 5% skimmed milk and probed with mouse anti-COX-IV antibody (Molecular Probes) or β-actin antibody (Sigma-Aldrich) as a loading control. Following incubation with horseradish peroxidase-conjugated anti-mouse secondary antibody (DAKOCytomation, Glostrup, Denmark), the bound antibody was visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
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