Samples of isolated LA were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5‐μm sections. Some sections were incubated with primary anti‐MCP‐1 or anti‐VCAM‐1 overnight at 4°C and then incubated with appropriate biotin‐conjugated secondary antibody (Vector Laboratories). Sections were visualized using a 3,3′‐Diaminobenzidine Substrates kit (Vector Laboratories) as immunostaining‐positive areas and counterstained with hematoxylin. Other sections were incubated with primary anti‐CD68 or anti‐MCP‐1 and anti‐collagen type 1 overnight at 4°C and then incubated with appropriate biotin‐conjugated secondary antibody (Vector Laboratories). These sections were visualized by immunofluorescence with fluorescein and cyanine 3–conjugated streptavidin and mounted with mounting medium using DAPI to visualize nuclei. Micrographs were digitized using a BZ‐9000 Biolevo Epifluorescence Microscope (Keyence). CD68‐positive cells in 3 images at magnification of ×400 were counted and averaged using imaging software (Keyence), as described.
Biotin conjugated secondary antibody
Manufactured by Vector Laboratories
Sourced in United States
Biotin-conjugated secondary antibodies are designed to detect and amplify the signal from primary antibodies in various immunoassays, such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISA). The biotin moiety covalently attached to the secondary antibody can be used to interact with streptavidin or avidin, enabling signal amplification and visualization of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc kit, by Vector Laboratories (5 mentions)
Streptavidin horseradish peroxidase, by Vector Laboratories (4 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (4 mentions)
Axiovert microscope, by Zeiss (4 mentions)
Abc kit, by Vector Laboratories (3 mentions)
Dab substrate, by Vector Laboratories (3 mentions)
Ab15580, by Abcam (3 mentions)
Ab16669, by Abcam (3 mentions)
Ab183685, by Abcam (3 mentions)
Sa 5704, by Vector Laboratories (3 mentions)
Cytoseal 60 mounting medium, by Thermo Fisher Scientific (3 mentions)
Eclipse 80i microscope, by Nikon (3 mentions)
Vectastain abc, by Vector Laboratories (2 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (2 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Vectastain abc reagent, by Vector Laboratories (2 mentions)
Anti cdk4, by Santa Cruz Biotechnology (2 mentions)
Ncl ki67, by Leica (2 mentions)
49 protocols using biotin conjugated secondary antibody
1
Immunohistochemical Analysis of Inflammatory Markers
2
Immunohistochemical Localization of CGRP
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We fixed lungs in formalin for 24 h before paraffin embedding. Paraffin blocks were sectioned at four-microns and stained with hematoxylin and eosin. Paraffin sections were cut and processed from xylene through a graded ethanol series (100%, 95% and 70%) to PBS. Unmasking was performed using microwave heating in sodium citrate buffer (0.01M at pH6.0). Endogenous peroxidases were blocked with 3.5% H2O2, and immunohistochemistry was performed with an overnight incubation with anti-CGRP antibody (Sigma C8198, 1:1000). Biotin-conjugated secondary antibodies (Vector Laboratories) were used at a dilution of 1/200 in blocking solution. After secondary antibody binding, detection was performed via a biotin-peroxidase complex (Vectastain ABC, Vector Laboratories) with DAB substrate (Vector Laboratories). Haematoxylin was used to counterstain.
3
Histological and Immunohistochemical Analysis of Skin Biopsies
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In the histological analysis, skin biopsies were fixed in 10% buffered formalin and embedded in paraffin and then 4-μm skin sections were cut and stained with hematoxylin and eosin (H&E) for cell counts and to measure epidermal or dermal thicknesses, as well as with o-toluidine blue for mast cell counts. In the immunohistochemical analysis, skin specimens were embedded in Tissue-Tek oxacalcitriol compound (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) and frozen quickly on dry ice. Staining of different subtypes of T-cells (CD3+, CD4+ and CD8+) was conducted as described earlier [19 (link)]. Briefly, the 4-μm skin samples were fixed with cold acetone and stained using the ChemMate (DakoCytomation, Glostrup, Denmark) staining kit. Primary anti-mouse monoclonal antibodies were obtained from BD Pharmingen (San Diego, CA). Macrophages (F4/80+) were stained in a similar technique with purified rat anti-mouse F4/80 antibody (clone BM4007S, Acris Antibodies, Herford, Germany). Biotin-conjugated secondary antibodies were obtained from Vector Laboratories Inc. (Burlingame, CA). The sections were examined under light microscopy (Leica DM 4000B, Wetzlar, Germany). Inflammatory cells were counted from 15 high-power fields (HPF) at 1000x magnification, mast cells, T-cell subtypes and macrophages at 400x magnification.
4
Immunohistochemical Analysis of Lung Tumors
5
Immunohistochemical Analysis of Bone Implants
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The presence of osteoblasts was detected by immunohistochemistry using an antibody specific for human osteocalcin (M184, Takara Bio Inc., Japan). To determine the origin of blood vessels in the re-vascularised bone implants, a dual immunofluorescence protocol using species-specific CD31 antibodies was performed to detect endothelial cells; human-specific CD31 (1:400, ab76533, Abcam, UK) and mouse-specific CD31 (1:400, ab56299, Abcam, UK). To establish whether genetic alterations observed between PDXs growing in the fat pad and those that had metastasised to bone were translated into changes in protein expression immunohistochemistry for IL-1B (1:200, ab2105, Abcam), IL1R1 (1:200, ab154524, Abcam), S100A4 (1:500, ab40722, Abcam, hRAS (1:200, ab97488, Abcam), DKK (1:200, Ab38594, Abcam), Gamma Catenin (1:25, 2309, Cell signalling), Fibronectin (1:50, ab32419, Abcam). Staining was visualised with corresponding biotin-conjugated secondary antibodies (Vector Laboratories, 1:200) and either avidin FITC (mouse) or avidin TRITC (human) (Vector Laboratories).
6
Immunohistochemical Analysis of Brain Development
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Immunohistochemical staining was carried out as described previously (Song et al., 2016 ). Briefly, brains at various stages were harvested after fixation with 4% paraformaldehyde (PFA) overnight and sectioned in a cryostat at 20 μm. The following primary antibodies were used: TLE4 (1:300, mouse; SC365406, Santa Cruz), BrdU (1:1,000, rat; OBT0030G, Accurate), and PH3 (rabbit; 06-570, Upstate). The sections were incubated with primary antibodies overnight at 4°C, followed either by biotin-conjugated secondary antibodies (Vector Laboratories) for 3 h and then Cy3-conjugated streptavidin or with secondary antibodies conjugated to Alexa fluorochromes (Molecular Probes, Invitrogen). All slides were mounted with 75% glycerol containing nuclei counterstaining, Hoechst 33258 (Sigma, St. Louis, MO, United States). Images were captured with an epifluorescence microscope (Eclipse 80i, Nikon) equipped with software NIS-Elements F400.
7
Immunohistochemical Staining of Breast Cancer
8
Immunohistochemical Characterization of Cells
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Fresh sections (formalin-fixed, paraffin embedded) were dewaxed following standard protocols and antigen retrieval with pH6 citrate retrieval buffer was undertaken for SMMHC, CK14, p63 and Ki67 staining. Sections were stained with anti-α-smooth muscle actin (1 μg/ml, Abcam, ab66133), anti-smooth muscle myosin heavy chain 1+2 (1 μg/ml, Abcam, ab124679), anti-Ki67 (1 μg/ml, Abcam, ab15580), anti-cytokeratin 14 (5 μg/ml, Abcam, ab53115), anti-p63 (0.75 μg/ml, Abcam, ab124762), or with isotype control antibodies, overnight at 4°C. Biotin-conjugated secondary antibodies (Vector Laboratories, CA, USA) were applied for 1 h and signal was amplified by conjugation of HRP (ABC kit, Vector Laboratories). Antibodies were visualized with DAB prior to nuclear counterstaining with hematoxylin.
9
Antibodies for Protein Detection
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The following antibodies were used: anti-HERC2 monoclonal (BD Biosciences); anti-HERC2 polyclonal [32 (link)]; anti-p21 (C-19); anti-p62 (SQSTM1 (D-3): sc-28359); anti β-actin (Santa Cruz Biotechnology, Inc.); anti-calbindin D-28k polyclonal (Cb-38a, Swant); anti-calbindin D-28k monoclonal (Cb-955, Sigma); anti-p53 Ab-5 (DO-7) (Neo Markers); anti-USP33 (Proteintech); anti-Ran [62 (link)]; anti α-tubulin (Ab-1, Calbiochem); Alexa Fluor® 488 donkey-anti-rabbit (A21207), and Alexa Fluor® 594 donkey-anti-mouse (A21203) (Invitrogen); horseradish peroxidase-conjugated secondary antibodies (Invitrogen); biotin-conjugated secondary antibodies (Vector); and the Avidine-Streptavidine Elite Kit (Vector).
10
Immunohistochemical Analysis of Alpha-Synuclein and p62
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At TEM, alpha-syn and p62 were also labeled by immuno-peroxidase. Cell pellets were fixed with 2.5% paraformaldehyde and 0.1 glutaraldehyde dissolved in PBS for 90 min. After washing in PBS, cell pellets were incubated in 0.002% hydrogen peroxide in 0.05 M Tris–HCl buffer, pH 7.6 for 2.5 min. Then, after washing in PBS, cell pellets were permeabilized in ethanol (10% for 5 min, 25% for 5 min, and 10% for 5 min) and pre-blocked with a solution containing 10% NGS and 0.2% saponin in PBS for 30 min. Samples were then incubated with anti-alpha-syn AbI (1:100, Abcam) or anti-p62 AbI (1:100, Abcam) diluted in 10% NGS and 0.2% saponin in PBS for 24 h. Then, samples were incubated with a solution containing the biotin-conjugated secondary antibodies (Vector) diluted 1:100 for 1 h at room temperature. After washing in PBS, samples were incubated in avidin–biotin peroxidase complex (Vector) for 1 h. After washing in PBS samples were incubated in 0.075% DAB (Vector) for a few minutes.
Samples were washed in PBS and osmicated, dehydrated, and embedded in Epoxin resin. Ultra-thin sections were cut by ultra-microtome (Leica Microsystems) and were observed under TEM (JEOL JEM SX100, JEOL, Tokyo, Japan).
Adobe Photoshop CS4 Extended program (version 11.0, Adobe Systems Inc., San Jose, CA, USA) was used to create the artwork.
Samples were washed in PBS and osmicated, dehydrated, and embedded in Epoxin resin. Ultra-thin sections were cut by ultra-microtome (Leica Microsystems) and were observed under TEM (JEOL JEM SX100, JEOL, Tokyo, Japan).
Adobe Photoshop CS4 Extended program (version 11.0, Adobe Systems Inc., San Jose, CA, USA) was used to create the artwork.
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