Skin samples were excised for protein analysis and homogenized in MENG buffer (20 mM MOPS, 2 mM EDTA, 0.02% sodium azide, 10% Glycerol, pH 7.4), supplemented with 20 mM molybdate, 500 mM NaCl, and cOmplete Mini protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany). Samples were resolved by 8% tricine-SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Rpt1 antibody was used to probe for AHR (Perdew et al. 1995 (link)) and B-actin was detected with B-actin (C4) antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Biotin-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) and 125I-streptavidin were used for detection and radioactivity was visualized using BioMax film. 125I-streptavidin was prepared as described previously (Narayanan et al. 2012 (link)).
Biotin conjugated secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States, Cameroon
Biotin-conjugated secondary antibodies are a type of laboratory reagent used in various immunological techniques. These antibodies are designed to bind specifically to primary antibodies, with the biotin molecule covalently attached to the secondary antibody. The biotin moiety can then be used to detect or isolate the target antigen-antibody complex, typically through interaction with streptavidin or avidin-based detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam hrc camera, by Zeiss (3 mentions)
Axio imager a1 microscope, by Zeiss (3 mentions)
Axiovision software, by Zeiss (3 mentions)
Avidin biotin complex, by Vector Laboratories (3 mentions)
Streptavidin alexa 488, by Thermo Fisher Scientific (2 mentions)
Anti pstat5tyr694 primary antibody, by Cell Signaling Technology (2 mentions)
Peroxidase conjugated streptavidin, by Jackson ImmunoResearch (2 mentions)
Complete mini protease inhibitors cocktail, by Roche (1 mentions)
Rat anti pecam1, by BD (1 mentions)
Confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Rabbit anti laminin, by Merck Group (1 mentions)
Cy3 or cy5, by Jackson ImmunoResearch (1 mentions)
Ab13970, by Abcam (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mca497, by Bio-Rad (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
2mod 1g6 as, by Euromedex (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
25 protocols using biotin conjugated secondary antibody
1
AHR Protein Analysis in Skin Samples
2
Immunoblotting Protein Detection Protocol
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Cell extracts were resolved on 8% tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis proteins were transferred to PVDF membrane. Proteins were detected using antibodies listed in online supplement (Table 1S ). Primary antibodies were visualized with species appropriate biotin-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) and a subsequent incubation with 125I-streptavidin.
3
Immunoblot Analysis of Membrane Proteins
4
Immunohistochemical Staining of OBSCs
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OBSCs were fixed for 20 min in 4% PFA in phosphate buffered saline (PBS), washed three times in PBS, then blocked for 1 h in PBS supplemented with 0.5% Triton X-100 and 3% normal Goat Serum (Sigma G9023). Slices were incubated at 4 °C in primary antibody (diluted in blocking solution) overnight. In order to detect PDGFRβ, heat-mediated antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) for 40 min at 80 °C prior to primary antibody incubation. Slices were washed a further three times in 0.5% Triton-X100 in PBS (PBS-TX) then incubated with secondary antibodies (Life Technologies and Jackson) (1:500 dilution in blocking solution for 2 h at 4 °C). Three final PBS-TX washes were conducted before slices were mounted on slides and images captured using a Leica Confocal Microscope. Primary antibodies used: rabbit anti-PDGFRβ (28E1) (1:200, Cell Signalling, Cat. No: 3169S), rat anti-PECAM-1 (1:400, BD, Cat. No: 550274), rabbit anti-laminin (1:200, Sigma, Cat. No: L9393), Mouse MOAB-2 (pan Aβ) (1:1000, Merck-Millipore, Cat. No: MABN254) Rabbit Ki67 (1:1000, Abcam, Cat. No: ab15580) secondary staining was conducted using species-specific fluorophore-conjugated (Streptavidin Alexa 488, Molecular Probes; Cy3 or Cy5, Jackson,) or biotin-conjugated secondary antibodies (Jackson). DAPI (1 μg/mL, Sigma) was used to counterstain nuclei.
5
Immunofluorescence and Immunohistochemistry Protocol
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Primary antibodies used include: anti-GFP (1/2,000, chicken polyclonal, Abcam ab13970); anti-Pax7 (1/20, mouse monoclonal, DSHB); anti-Calcitonin receptor (1/100, rabbit polyclonal, Serotec AHP 635); anti-HP1γ (1/2,000, mouse monoclonal, Euromedex, 2MOD-1G6-AS); anti-Myosin Heavy Chain (1/200, rabbit polyclonal, kindly provided by G. Cossu); anti-Ki67 (1/300, rabbit polyclonal, Abcam 15,580); anti-p53 (1/500, rabbit polyclonal, Leica, MC5); anti-p21 (1/2, rat monoclonal, CNIO HUGO291); anti-Tcf4 (1/100, rabbit monoclonal, Cell Signalling C48H11); anti-F4/80 (1/50, rat monoclonal, Serotec MCA 497). Alexa-conjugated secondary antibodies (1/500, Molecular Probes) together with 1 μg ml−1 of Hoechst-33342 were used for immunofluorescence. Following immunofluorescence, cells and sections were mounted with VECTASHIELD Mounting Media between slide and coverslip. Immunohistochemistry was performed by incubation with Biotin-conjugated secondary antibodies (1/500, Jackson laboratories), followed by incubation with Peroxidase (HRP) Polymer conjugated streptavidin (1/2,000, S2438 Sigma). Peroxidase activity was detected using the DAB Peroxidase Substrate Kit (SK-4100 Vector).
6
Immunohistochemical Analysis of Murine Stroke Model
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One day after permanent middle cerebral occlusion, mice were transcardially perfused with PBS followed by 4% paraformaldehyde and sections prepared as previously described.8 (link) The primary antibodies used were rabbit anti-AQP4 (aquaporin 4; 1:1000, Millipore), chicken anti-GFP (green fluorescent protein; 1:1000, Abcam), rabbit anti-claudin 5 (1:1000, Abcam), mouse anti-NeuN (neuronal nuclei; 1:400, Millipore), rabbit anti-neural/glial antigen 2 (NG2; 1:200, Millipore), rabbit anti–platelet-derived growth factor receptor (PDGFR)-β (1:200, Cell Signaling), anti–platatelet endothelial cell adhesion molecule-1 (CD31; 1:400, BD), rabbit antivascular endothelial-cadherin (1:1000, Abcam), rabbit anti–ZO-1 (zonula occludens 1; 1:500, Fisher), and staining revealed using species-specific fluorophore-conjugated (Streptavidin Alexa 488, Molecular Probes; Cy3 or Cy5, Jackson) or biotin-conjugated secondary antibodies (Jackson). Biotinylated secondary antibodies were revealed using the ABC kit (Vector labs). 4’,6-diamidine-2’-phenylindole (1 μg/mL, Sigma) was used for counterstaining.
7
Immunohistochemical Staining of Enteroids
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Paraffin-embedded enteroids, intestinal tissue, and tissue microarrays were incubated at 56°C prior to de-waxing and rehydration. Antigens were retrieved by boiling sections in 10 mM citric acid, pH 6.0, for 2 hrs. Samples were blocked in 1% BSA, 0.3% Triton-X-100, and 10% normal goat serum for 1 hr. Endogenous peroxidases were quenched in 3% hydrogen peroxide for 5 minutes. In conjunction with biotin-conjugated secondary antibodies (Jackson ImmunoResearch, diluted 1:200) stains were developed with the VECTASTAIN Elite ABC Kit (Vector Laboratories, cat# PK-6100) and the DAB Peroxidase (HRP) Substrate Kit (Vector Laboratories, cat# SK-4100). Tissues were dehydrated and cover-slipped with Cytoseal (Thermo Scientific, cat# 8310–4). Primary antibodies used for IHC were anti-ARID3A (1:100, ProteinTech, Chicago IL, cat# 14068-1-AP), anti-β-catenin [D10A8] XP Rabbit mAb (1:100, Cell Signaling, Danvers MA, cat# 8480), rabbit anti-HIF3A antibody (1:200, Sigma-Aldrich, St. Louis MO, cat# SAB2900410), anti-HMGA1 antibody [EPR7839] (1:250, Abcam, Cambridge MA, cat# ab129153), and anti-HMGA2 [D1A7] rabbit mAb (1:400, Cell Signaling, Danvers MA, cat# 8179).
8
Western Blot Analysis of Protein Expression
9
Immunostaining Analysis of Tau and Microglial Markers
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Immunostaining analysis was carried out as described26 (link),40 (link). The primary antibodies used were cis P-tau mAb (clone #25), hyperphosphorylated tau epitopes antibodies (AT8 and AT100) (Innogenetics), microglia-specific antibody (Iba1) (Wako), and neuonal nuclei specific antibody (NeuN) (Milipore). After treatment with 0.3% hydrogen peroxide, slides were briefly boiled in 10 mM sodium citrate, pH 6.0, for antigen enhancement. The sections were incubated with primary antibodies overnight at 4 °C. Then, biotin-conjugated secondary antibodies (Jackson ImmunoResearch) were used to enhance the signals. Manufacturer-supplied blocking buffer (Invitrogen) was used for each reaction. The sections were washed four times with TBS after each step. Labeled sections were visualized with a Zeiss confocal microscope. The gain of confocal laser was set at the level where there are no fluorescence signals including autofluorescence in sections without primary antibody but with secondary antibody (Fig. S10 ). Slides were imaged on a Leica SPE microscope (Leica Microsystems, Wetzlar, Germany) and VS120 slide scanner (Center Valley, PA). Immunostaining images and their co-localization were quantified using Fiji/ImageJ Coloc 2.
10
Nuclear and Cytosolic Protein Extraction
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Cytosolic and nuclear extracts were prepared as previously described [18 (link)]. Briefly, after 1 h ligand treatment, cells were washed and scraped into PBS. Cell pellets were resuspended in MENG (25 mM MOPS, 2 mM EDTA, 0.02% sodium azide, and 10% glycerol, pH 7.4) containing protease inhibitor cocktail (Roche) and homogenized with a stainless-steel Dura-Grind Dounce homogenizer (Wheaton Instruments, Millville, NJ, USA). Cell homogenates were centrifuged at 1000× g for 20 min, and the supernatant was then subjected to ultracentrifugation to generate cytosol. The nuclear pellet was washed three times with homogenization buffer, and then extracted with MENG containing 500 mM NaCl for 1 h, followed by centrifugation. Cell extracts were resolved on 8% tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to PVDF membrane. Specific proteins were detected using anti-AHR (Thermo Fisher Scientific, Inc., Waltham, MA, USA), or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies, and visualized with species appropriate biotin-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) and a subsequent incubation with 125I-streptavidin, which was generated as described [69 (link)].
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