Proteins were resolved by 10 % polyacrylamide gel electrophoresis (PAGE) under reducing conditions and visualized by silver staining, performed using the Silver Stain Kit (BioRad, Hercules, CA, USA) according to the manufacturer's instructions. Proteins separated on 10 % polyacrylamide gel were transferred to nitrocellulose membrane by Western blotting. Non-specific binding was blocked by TBS (50 mM Tris-HCl, 150 mM NaCl) pH 7.6/1 % BSA for 1 h at room temperature. The membranes were incubated overnight at 4 °C with rabbit anti-human galectin-1 (1:3000, INEP) and goat anti-mouse galectin-1 affinity-purified polyclonal antibody (1:1000, R&D, Minneapolis, MN, USA), followed by secondary antibody-HRP (biotin-labeled goat anti-rabbit IgG, Vector Laboratories, 1:3000; Rabbit anti-Goat IgG (H+L), HRP, Invitrogen, Waltham, MA, USA, 1:10000). Non-specific binding was assessed by incubation with secondary antibody only. Protein bands were detected by ABC (in case of biotinylated secondary antibody) or by adding 0.05 % 3.3'-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories).
Biotin labeled goat anti rabbit igg
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
Biotin-labeled goat anti-rabbit IgG is a secondary antibody reagent used in immunoassays and other immunochemical techniques. It is produced by immunizing goats with rabbit immunoglobulin G (IgG) and then conjugating the resulting antibodies with biotin.
Automatically generated - may contain errors
Lab products found in correlation
Silver stain kit, by Bio-Rad (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Vector Laboratories (1 mentions)
Ha tcp ceramic powder, by Zimmer Biomet (1 mentions)
Ti u inverted microscope, by Nikon (1 mentions)
Ds fi1 digital camera, by Nikon (1 mentions)
Nis elements basic research acquisition and analysis software package, by Nikon (1 mentions)
Permount mounting medium, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (1 mentions)
Pecam 1, by Santa Cruz Biotechnology (1 mentions)
Sirt2, by Santa Cruz Biotechnology (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Nampt, by Fortis Life Sciences (1 mentions)
Collagen type 4, by Merck Group (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
5 protocols using biotin labeled goat anti rabbit igg
1
Protein Characterization by Western Blot
2
Bone Regeneration with HA/TCP and BMP4
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The primary cultured mouse bone‐derived cells (1 × 106) were mixed with 100‐mg hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer, Warsaw, IN, USA) alone or with BMP4 (5 μg; Peprotch, Rocky Hill, NJ, USA) in a 0.5% fibrin gel, and then transplanted s.c. into immunocompromised mice (NIH‐bg‐ nu‐xid; Harlan Laboratories, Indianapolis, IN, USA) for 6 and 12 weeks.
For histomorphometric analysis of newly formed mineralized tissue, samples were harvested and fixed in 4% PFA, decalcified in 10% EDTA (pH 7.4), embedded in paraffin, and stained with H&E, Masson's trichrome (Polysciences Inc., Warrington, PA, USA), or processed for immunohistochemistry. For immunohistochemistry, proteins were detected with anti‐BSP(17 ) at a dilution of 1:100 as the primary antibody and a biotin‐labeled goat anti‐rabbit IgG (Vector Labs) as the secondary antibody. Tartrate‐resistant acid phosphatase staining was performed. The total mineralized area among the regenerated bone‐ and marrow‐like tissue was analyzed using the LS starter program (Olympus Soft Imaging Solutions).
For histomorphometric analysis of newly formed mineralized tissue, samples were harvested and fixed in 4% PFA, decalcified in 10% EDTA (pH 7.4), embedded in paraffin, and stained with H&E, Masson's trichrome (Polysciences Inc., Warrington, PA, USA), or processed for immunohistochemistry. For immunohistochemistry, proteins were detected with anti‐BSP(
3
Immunoperoxidase Staining for 4-HNE Detection
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For immunoperoxidase staining, permeabilized sections were first treated with 1 % hydrogen peroxide in methanol to block endogenous tissue peroxidase, and then blocked in 2 % NGS. Slides were washed, incubated with polyclonal rabbit anti-4-hydroxynonenal (4-HNE, 1:250, Abcam) for 1 h at room temperature, washed again, and then treated with biotin-labeled goat anti-rabbit IgG (Vector Laboratories) at a 1:200 dilution for another hour at room temperature. These steps were followed by sequential incubations with avidin-DH-biotin complex (Vector Laboratories) and then 0.5 mg/ml 3,3′-diaminobenzidine (Sigma-Aldrich) in PBS containing 0.01 % hydrogen peroxide. All slides were counterstained with hematoxylin and mounted with coverslips using Permount mounting medium (Thermo Fisher Scientific) for light microscopy. Slides were imaged using a Nikon Ti-U inverted microscope equipped with a Nikon DS-Fi-1 digital camera and supported by the NIS-Elements Basic Research acquisition and analysis software package (Nikon Instruments Inc.).
4
Immunohistochemical Analysis of Steroidogenic Enzymes
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Antibodies against CD31 (PECAM-1) were purchased from Santa Cruz Biotechnology Inc. (sc-1506; Dallas, TX); antibodies against Caspase-3 (Casp-3) from Cell Signaling Technologies (D175 5A1E; Beverly, MA); antibodies against α-Smooth Muscle Actin (αSMA) and cholesterol side chain cleavage enzyme (P450scc) were purchased from Abcam Inc. (ab5694-100, Cambridge, UK) and Chemicon Inc. (AB1294, Billerica, MA), respectively. CD31 produces a membranal stain, while Casp-3, αSMA, and P450scc produce cytoplasmic stains. Biotin-labeled goat anti-rabbit IgG was purchased from Vector Lab (BA-1000, Burlingame, CA) and streptavidin-horseradish peroxidase (HRP, 550946) conjugate from BD Biosciences (San Jose, CA). Pregnant Mare Serum’s Gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were purchased from Sigma Chemical Co. (St. Louis, MO). EDN2 peptide was purchased from American Peptide Co., Inc. (Sunnyvale, CA). Other common molecular reagents were purchased from Invitrogen Life Technologies, Inc (Carlsbad, CA).
5
Histological Analysis of Kidney Tissue
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The kidney tissues were fixed in 10% neutral-buffered formaldehyde, embedded in paraffin and sectioned at 4-mm thickness. The sections were stained by the periodic acid Schiff, periodic acid-methenamine silver (PAM), or Masson-trichrome methods for light microscopic analysis and morphometry. Immunohistochemistry was performed as described previously (Hasegawa et al., 2010) . Briefly, 4-mm-thick paraffin sections were fixed in 3% formaldehyde and stained with primary antibodies against FLAG (Sigma-Aldrich), Nampt (Bethyl Laboratories, Montgomery, TX, USA), Sirt1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Sirt2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Sirt3 (Cell Signaling Technology, MA, USA), Sirt6 (LifeSpan BioSciences, WA, USA) or type-IV collagen (Millipore, Bedford, MA, USA). The sections were stained with biotin-labeled goat anti-rabbit IgG (Vector Laboratories, UK) or biotin-labeled anti-mouse IgG (Vector) and then treated with the Vectastain Elite ABC Kit (Vector). Each photograph of the stained sections was scanned using a 3CCD camera (Olympus Optical, Tokyo, Japan). For morphometric analysis in the tubulointerstitium, the positive area for type-IV collagen staining in the interstitium per high-power field was measured in a blinded manner.
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