Crude lung CM harvested after 2 days of incubation was concentrated 60‐fold, boiled in reducing Laemmli buffer, and applied to an 8% SDS‐PAGE gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane and incubated with a mouse polyclonal antibody against α3 laminin chain (Abnova, Taipei, Taiwan) diluted 1:500, in blotto buffer, and monoclonal antibodies against β3 laminin chain (clone 17/kalinin B1 Transduction Laboratories, Lexington KY) diluted 1:1000 at 0.25 μg/mL and γ2 laminin chain (clone D4B5 Millipore) diluted 1:800–2 μg/mL. Where applicable, β actin Abcam8226 (Abcam, Cambridge, MA) was diluted 1:1000–23 μg/mL. After incubation, washing and application of a biotinylated goat anti‐mouse secondary antibody (Thermo‐Fisher Scientific) diluted 1000 times (0.8 mg/μL), the signals were developed with the Pico Super Signal West chemoluminescent detection system (Thermo‐Fisher Scientific) according to the manufacturer's instructions and bands were visualized with the C‐Digit scanner (Licor, Lincoln, NE).
Secondary biotinylated goat anti mouse antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Secondary biotinylated goat anti-mouse antibody is a laboratory reagent used for detection of mouse primary antibodies in various immunoassay techniques. It provides a biotinylated secondary antibody that can bind to the primary mouse antibody, allowing for amplification and visualization of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Thermo Fisher Scientific (3 mentions)
Diaminobenzidine solution, by Thermo Fisher Scientific (2 mentions)
C digit scanner, by LI COR (1 mentions)
Adrenaline, by Merck Group (1 mentions)
Mmp13, by Abcam (1 mentions)
Dna extraction kit, by Merck Group (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Citrate buffer, by Solarbio (1 mentions)
Ptch1, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
5 protocols using secondary biotinylated goat anti mouse antibody
1
Laminin Subunit Expression in Lung CM
2
Rotenone-Induced Parkinson's Disease Model
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Rotenone was procured from TCI (Plau, USA) and olive oil from Thuruthel Drug Lines, (ERUMBA. Pharmacy Payyannur Kerala, India). FRAP (ferric reducing antioxidant power) was purchased from SD fine chemicals, India, TPTZ (2,4,6-Tri-(2-pyridyl)-5triazine) and Adrenaline were procured from Sigma Aldrich, India, anti-mouse alpha Synuclein primary antibody and biotinylated goat anti-mouse secondary antibody from Thermo Fisher Scientific, USA. Embelin was isolated as per the procedure in the Central Research Laboratory (CRL) facility of Department of Research and Development, Saveetha Institute of Medical and Technical Sciences, Chennai, India. Levodopa was obtained from SR Laboratories (Maharashtra, India) dimethyl sulfoxide (DMSO) from Pure Chemicals and hydroxypropyl cellulose (HPC) from HIMEDIA, (India). All the other chemicals and reagents used were of analytical grade.
3
Cartilage Extracellular Matrix Regulation
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DNA extraction kit and Tamoxifen were purchased from Sigma–Aldrich (Sigma-Aldrich, St. Louis, MO, United States). 4% paraformaldehyde, 14% elhylene diamine tetraacetic acid (EDTA) and citrate buffer were purchased from Solarbio (Solarbio, Beijing, China). Normal goat serum, secondary biotinylated goat anti-mouse antibody and diaminobenzidine (DAB) solution were obtained from Invitrogen (Invitrogen, Frederick, MD, United States). COL-2, MMP-13 and pSMAD-2 primary antibodies were brought from Abcam (Abcam, Cambridge, MA, United States).
4
Immunohistochemical Analysis of Hedgehog Signaling
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After deparaffinized, sections were treated with 0.3% hydrogen peroxide to reduce endogenous peroxidase activity. Then antigen retrieval was performed with the sections incubated at 60°C with 0.1 mol/L citrate buffer overnight. Normal goat serum (diluted 1:20) (Invitrogen, MD, USA) was added for 30 min at room temperature to block non-specific staining. Subsequently, the sections were treated with primary antibodies, including Gli2 (1:100), Gli3 (1:100), Smo (1:100), Ptch1 (1:100), Ihh (1:100) (Abcam, MA, USA) and further incubated overnight at 4°C. Secondary biotinylated goat anti-mouse antibody (diluted 1:1000) (Invitrogen, MD, USA) was subsequently added for 30 min incubation the following day. Diaminobenzidine solution (Invitrogen, MD, USA) and followed by counterstaining with hematoxylin was used to detect positive staining of sections. The H-score ranged from 0 to 300 was used for semiquantitative assessment of IHC images according to both the intensity of staining and the percentage of cells stained.
5
Immunohistochemical Analysis of Cartilage Markers
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Three micrometre deparaffinized sections were treated with 0.3% hydrogen peroxide to reduce endogenous peroxidase activity. Then the sections were incubated for 20 min at 95 °C with 0.1 mol/L citrate buffer as antigen retrieval. Non-specific staining was blocked by incubation of normal goat serum (diluted 1:20) (Invitrogen, MD, USA) for 20 min at room temperature. Subsequently, the sections were treated with collagen II (COL2) (diluted 1:1000), matrix metallopeptidase 13 (MMP13) (diluted 1:100), transforming growth factor beta receptor II (TGFBR2) (diluted 1:100) or phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD2) (diluted 1:100) primary antibodies (Abcam, MA, USA) and further incubated overnight at 4 °C. Secondary biotinylated goat anti-mouse antibody (diluted 1:1000) (Invitrogen, MD, USA) was added for 30 min on the second day. Positive staining of sections was detected by diaminobenzidine solution (Invitrogen, MD, USA) followed by counterstaining with hematoxylin. The primary antibody was not added to negative controls.
The nuclei were considered positive for TGFBR2, pSMAD2 and MMP13 labeling if their immunostains were equal or larger than 50% of the nuclear area. Weak brown stains were excluded from the counting. The rate of TGFBR2, pSMAD2 and MMP13 positive cells were determined using Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, USA).
The nuclei were considered positive for TGFBR2, pSMAD2 and MMP13 labeling if their immunostains were equal or larger than 50% of the nuclear area. Weak brown stains were excluded from the counting. The rate of TGFBR2, pSMAD2 and MMP13 positive cells were determined using Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, USA).
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