Postfixed cryostat sections in acetone (at 4°C for 5 minutes) were used. For vascular endothelial growth factor (VEGF) and matrix MMP 9, CAT, and SOD, the following primary antibodies were used: rabbit polyclonal anti-SOD-1 (Abcam), rabbit polyclonal anti-CAT (Abcam), mouse monoclonal anti-VEGF (Abcam), and goat polyclonal anti-MMP 9 (Santa Cruz Biotechnology, Santa Cruz, California, USA). The binding of the primary antibodies was demonstrated using the HRP/DAB UltraVision Detection System, as described previously (6,7). For secondary antibodies, biotinylated goat anti-rabbit IgG, biotinylated goat anti-mouse IgG, or donkey anti-goat IgG (Thermo Fisher Scientific) (10 minutes) were employed. Cryostat sections in which the primary antibodies were omitted from the incubation media, served as negative controls. The sections for VEGF and MMP9 were counterstained with Mayer's hematoxylin. After the staining procedure, the samples were immediately examined using an Orthoplan Leitz light microscope, equipped with a Leica DC 500 digital camera.
Biotinylated goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Biotinylated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG). The biotin molecule attached to the antibody allows for detection and signal amplification in various immunoassays and detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Ecl advance blocking agent, by GE Healthcare (2 mentions)
Biotinylated goat anti human igg, by Agilent Technologies (2 mentions)
Donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Biotinylated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti vegf, by Abcam (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Westernbright quantum hrp substrate, by Advansta (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Complete protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Extravidin peroxidase, by Merck Group (1 mentions)
Ultravision quanto detection system hrp dab kit, by Thermo Fisher Scientific (1 mentions)
Anti vegf, by Merck Group (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti mbp, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 635 streptavidin, by Thermo Fisher Scientific (1 mentions)
Streptavidin horseradish peroxidase, by R&D Systems (1 mentions)
Infinite f200 pro spectrophotometer, by Tecan (1 mentions)
9 protocols using biotinylated goat anti mouse igg
1
Immunohistochemical Analysis of Angiogenic Factors
2
Western Blot Analysis of Proteins in CSF
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Five microlitre CSF was diluted in 15 μl PBS containing a complete protease inhibitor (Thermo Scientific). The samples were denatured in 5× Laemmli buffer, separated by SDS–PAGE gel electrophoresis, and transferred to the nitrocellulose membrane. Following blocking with Odyssey Blocking buffer (LI‐COR Biosciences), the membrane was first incubated with biotinylated goat anti‐mouse IgG (1:2,000; Thermo Scientific) and then with HRP‐conjugated Streptavidin (1:5,000; Thermo Scientific). Protein bands were detected and quantified by WesternBright Quantum HRP substrate (Advansta) in Amersham Imager 600 (GE Healthcare).
3
Immunohistochemical Analysis of Amyloid-Beta in 5xFAD Mouse Brain
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Mouse brain slices were pre-treated with 60% methanol, 1% H2O2 (30 min), followed by washes in 0.1 M TBS (pH 7.4), and blocked in TBS containing 0.3% TritonX-100 and 5% normal goat serum for 30 min to reduce unspecific binding of antibodies. Incubation with primary antibody was performed in blocking buffer over night at 4 °C.
For staining of untreated 5xFAD mouse brain, slices were incubated with 2 μg/ml K11 or commercially available antibody 6E10 (1:500, Biolegend, San Diego), followed by application of biotinylated goat anti-mouse IgG (Thermo Fisher Scientific).
When analyzing 5xFAD brain slices, treated with K11_IgG2a, 3D6_IgG2a, or IgG2a isotype control, samples were incubated with 2 μg/ml isoD7-Aβ specific Ab K16 (isotype IgG1), 6E10 (isotype IgG1) or 0.5 μg/ml 3D6 (isotype IgG2b). Then slices were incubated with biotinylated anti-mouse IgG1 or anti-mouse IgG2b (1:1000 Dianova, Hamburg) in TBS with 2% (v/v) BSA for 1 h followed by incubation with ExtrAvidin-peroxidase (Sigma-Aldrich, 1:1000) in the same buffer. After 3 washing steps (5 min in TBS), immunostaining was performed by treatment of sections with 0.05% (w/v) of the chromogenic substrate 3,3′-diaminobenzidin (DAB) in 0.05 M Tris, pH 7.6, with 0.015% (v/v) H2O2 for 4 min.
For staining of untreated 5xFAD mouse brain, slices were incubated with 2 μg/ml K11 or commercially available antibody 6E10 (1:500, Biolegend, San Diego), followed by application of biotinylated goat anti-mouse IgG (Thermo Fisher Scientific).
When analyzing 5xFAD brain slices, treated with K11_IgG2a, 3D6_IgG2a, or IgG2a isotype control, samples were incubated with 2 μg/ml isoD7-Aβ specific Ab K16 (isotype IgG1), 6E10 (isotype IgG1) or 0.5 μg/ml 3D6 (isotype IgG2b). Then slices were incubated with biotinylated anti-mouse IgG1 or anti-mouse IgG2b (1:1000 Dianova, Hamburg) in TBS with 2% (v/v) BSA for 1 h followed by incubation with ExtrAvidin-peroxidase (Sigma-Aldrich, 1:1000) in the same buffer. After 3 washing steps (5 min in TBS), immunostaining was performed by treatment of sections with 0.05% (w/v) of the chromogenic substrate 3,3′-diaminobenzidin (DAB) in 0.05 M Tris, pH 7.6, with 0.015% (v/v) H2O2 for 4 min.
4
Immunohistochemical Analysis of Brain Tissue
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Brain samples were harvested and kept in 4% paraformaldehyde for 24 h and immersed in 30% sucrose for 3-4 days at 4°C. After cryoprotection in 30% sucrose, the brains were rapidly frozen in isopentane and stored at −80°C. Twenty micrometer cryostat sections at the level of the thalamus (bregma −3.3 mm) according to a stereotaxic atlas [15 ] were processed for immunohistochemistry. Immunohistochemistry analyses were performed with the UltraVision Quanto Detection System HRP DAB kit (Thermo Fisher Scientific, USA). The primary antibodies utilized were rabbit anti-GFAP antibody (glial fibrillary acidic protein, Santa Cruz Biotechnology, USA, 1 : 200 dilution), anti-VEGF (vascular endothelial growth factor, Millipore Corporation, USA, 1 : 400), and anti-MBP (myelin basic protein, Santa Cruz Biotechnology, USA, 1 : 200). The secondary antibodies utilized were biotinylated goat anti-mouse IgG and goat anti-rabbit IgG (Thermo Fisher Scientific, USA, 1 : 400 dilution). The results were observed using the Leica vertical microscope.
5
Immunohistochemical Labeling of Drosophila Brains
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The fly brains were dissected in phosphate-buffered saline (PBS) solution and immediately transferred to 4% paraformaldehyde (PFA) for fixation for 20 min. Fixed brain samples were incubated in penetration and blocking buffer (PBS containing 2% Triton X-100 and 10% normal goat serum) for 2 h. During the penetration and blocking period, the brain samples were subjected to a degassing procedure. Brains were then incubated in the dilution buffer (PBS containing 0.25% Triton X-100, 1% normal goat serum) containing mouse 4F3 anti-discs large (DLG) monoclonal antibody (1:10, AB 528203, Developmental Studies Hybridoma Bank, University of Iowa) at 25°C for 24 h. After incubation, the tissues were washed in PBS containing 1% Triton X-100 (PBS-T) three times (each time 15 min), and incubated with biotinylated goat anti-mouse IgG (1:200, 31,800, Thermo Fisher Scientific) at 25°C for 24 h. Next, the brain samples were washed and incubated with Alexa Fluor 635 streptavidin (1:500, S32364, Thermo Fisher Scientific) at 25°C for 24 h. After intensive washing in PBS-T for three times, the brain samples were cleared and mounted in FocusClear (FC-101, CelExplorer).
6
Comparative Sensitivity Analysis of Amyloid-Beta Antibodies
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The sensitivity of the antibody 82E1 and the biosimilar Bapineuzumab antibody was further analysed by urea-bicine/bis-tris/Tris/sulphate sodium dodecyl sulphate (SDS)-PAGE followed by western immunoblotting. Stock solutions of synthetic Aβ1–40 were prepared in sample buffer (0.36 M bis-tris, 0.16 M bicine, 15% wt/vol sucrose, 1% wt/vol SDS and 0.0075% wt/vol bromophenol blue) and stored at −80°C. Different amounts of Aβ1–40 peptides (25–100 pg and 25–100 ng respectively) were loaded and separated by urea-bicine/bis-tris/Tris/sulphate SDS-PAGE and analysed by immunoblotting as previously described [40 (link)]. The blotting membranes were blocked in 2% enhanced chemiluminescence (ECL) advance blocking agent (GE Healthcare Life Sciences, Little Chalfont, UK) in phosphate-buffered saline (PBS) with 0.075% Tween 20 (PBS-T) for 1h at room temperature and probed with the primary antibody 82E1 or the biosimilar Bapineuzumab antibody (0.5 μg/μl in PBS-T) at 4°C overnight. After three washing steps with PBS-T, biotinylated goat anti-mouse IgG (1:3000 in PBS-T; Life Technologies, Carlsbad, CA, USA) for 82E1 and biotinylated goat anti-human IgG (DAKO, Glostrup, Denmark, 1:3000) for Bapineuzumab, followed by streptavidin-HRP, were employed as secondary reagents. Chemiluminescent signals obtained with ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) were recorded.
7
Comparative Sensitivity Analysis of Amyloid-Beta Antibodies
8
Quantifying DNA Damage by ELISA
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CPDs were measured by ELISA. Epidermal sheets were separated by NH4SCN as above. Epidermal DNA was isolated using a DNeasy Blood and Tissue kit (Qiagen) and denatured at 100 °C for 10 min. DNA (10 ng) was dried onto protamine sulfate-coated 96-well plates (Nunc), and ELISAs were performed using anti-CPDs (Cosmo Bio), biotinylated goat anti-mouse IgG (Invitrogen), streptavidin–horseradish peroxidase (R&D) and a TMB Substrate Reagent set (BD), as per the anti-CPDs manufacturer’s protocol. Absorbance was measured using an Infinite F200 Pro spectrophotometer (Tecan).
9
BrdU-Positive Cell Quantification
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To detect BrdU-positive cells, the DNA was denatured by 2N HCl, and the sections were incubated with a mouse anti-BrdU primary antibody (Thermo, 1:1000) for 48 h at 4°C. The slides were then washed in 0.1 M phosphate buffer 3 times and incubated with biotinylated goat anti-mouse IgG (Invitrogen, 1:3000) at 4°C overnight. Using an ABC kit (Vector Laboratories, USA), BrdU-positive cells were visualized and counted blindly with a microscope (Olympus, Japan).
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