Biotinylated secondary antibody

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Biotinylated secondary antibody is a laboratory reagent used in various immunoassay techniques. It serves as a detection tool, binding to the primary antibody and allowing for further signal amplification or visualization.

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73 protocols using biotinylated secondary antibody

Immunohistochemical Staining of Ctβ Protein

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A series of sections was thoroughly rinsed in 0.1M SPB and immersed in 1% aqueous sodium borohydride for 15 min followed by thorough rinsing in 0.1M SPB. The sections were then incubated overnight in 0.1M SPB containing 0.1% Triton X-100 (SPB-t) and anti-Ctβ antibody (see Primary Antibodies section below) was used at a dilution of 1:5000. The following day, sections were rinsed thoroughly in SPB-t and placed for one hour in SPB-t containing biotinylated secondary antibody (Jackson Laboratories, West Grove, PA) raised in donkey against goat at a dilution of 1: 200. After thorough rinsing, sections were placed for one hour in SPB-t containing avidin-biotin-peroxidase complex (ABC, Vector Laboratories, Burlingame, CA) used at 1:200, rinsed again in SPB and incubated for 5–10 min in a solution of 0.025M Tris buffer (pH 8.0) containing 0.015% 3,3′-diaminobenzidine (DAB), 0.4% nickel ammonium sulfate, and 0.0003% hydrogen peroxide (NiDAB), which generates an insoluble black reaction product. After further rinsing, the sections were mounted in rostrocaudal sequence on gelled slides and coverslipped under Permount (Fisher, St Louis, MO).

Yetnikoff L., Cheng A.Y., Lavezzi H.N., Parsley K.P, & Zahm D.S. (2015). Sources of input to the rostromedial tegmental nucleus, ventral tegmental area and lateral habenula compared: a study in rat. The Journal of comparative neurology, 523(16), 2426-2456.

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Immunostaining of Macrophages and BAT

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Macrophage and BAT immunostainings were performed on 8 µm-thick frozen sections using anti-F4/80 and anti-UCP-1 antibodies, respectively. Briefly, endogenous peroxidases were blocked with hydrogen peroxide on −20°C acetone fixed slices. Following a blocking step at room temperature, the primary antibody was applied on the tissue sections overnight at 4°C (anti-F4/80, 1∶200, 14-4801-85 (BM8), eBioscience; and anti-UCP-1, 1∶500, sc-6528 (C17), Santa Cruz Biotechnology). The appropriate biotinylated secondary antibody (Jackson ImmunoResearch) was incubated (1 h at RT) prior to revelation with serial incubation of ABC and DAB (Vector Labs) for UCP-1 staining and Simple Stain Mouse MAX PO (rat) kit (N-Histofine) for macrophage staining and counterstaining with hematoxylin was then performed.

Toczek J., Broisat A., Perret P., Desruet M.D., Fagret D., Riou L.M, & Ghezzi C. (2014). Periaortic Brown Adipose Tissue as a Major Determinant of [18F]-Fluorodeoxyglucose Vascular Uptake in Atherosclerosis-Prone, ApoE−/− Mice. PLoS ONE, 9(7), e99441.

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Immunohistochemistry and Western Blot Analysis of Neuroinflammatory Markers

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Antibodies for immunohistochemistry to detect Iba1 (goat polyclonal; catalog number ab5976), vimentin (rabbit monoclonal; clone EPR3776; catalog number ab92547), and CD68 (rabbit polyclonal; catalog number ab125212) were from Abcam (Cambridge, UK), that for detecting glial fibrillary acidic protein (GFAP; rabbit polyclonal; catalog number G4546) was from Sigma-Aldrich (St Louis, Mo) and for NeuN (mouse monoclonal; clone A60; catalog number MAB377) was from Millipore-Chemicon (Merck, Darmstadt, Germany). Alexa-fluor-conjugated secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). For additional analysis of morphological changes in Iba1-immunolabeled cells, the antibody to detect Iba1 was from Wako Pure Chemical (Osaka, Japan; catalog #019-19741) and the biotinylated secondary antibody was from Jackson ImmunoResearch (West Grove, PA, USA; catalog #711-005-152).
For Western blot analysis, antibodies to detect GFAP (rabbit polyclonal; catalog number G3893) and actin (rabbit polyclonal; catalog number A2066) were from Sigma-Aldrich; those for vimentin (rabbit monoclonal; catalog number ab92547) were from Abcam and for neurocan (mouse monoclonal; clone 650.24; catalog number MAB5212) were from Millipore-Chemicon. A peroxidase donkey anti-rabbit IgG (catalog number: 711-035-712) from Jackson ImmunoResearch was used as secondary antibody.

Yew W.P., Djukic N.D., Jayaseelan J.S., Walker F.R., Roos K.A., Chataway T.K., Muyderman H, & Sims N.R. (2019). Early treatment with minocycline following stroke in rats improves functional recovery and differentially modifies responses of peri-infarct microglia and astrocytes. Journal of Neuroinflammation, 16, 6.

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Visualizing Lineage Tracing Markers

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To visualize LacZ⁺ clones in whole mount, β-galactosidase (β-gal) activity staining was performed as previously described (Torres, 1998 (link)). Embryos were post-fixed in 4% paraformaldehyde (PFA).
To visualize EYFP⁺ clones, hearts went through a two-step process as follow:
-Whole mount immuno-staining (common protocol for immuno-histochemistry and immuno-fluorescence): Hearts were permeabilized using 0.5% Triton X-100 in PBS ON at 4°C. Hearts went through a process of endogenous peroxidase quenching using 0.03% hydrogen peroxide (H₂O₂) in PBS at room temperature (RT). Hearts were then blocked in PBST (PBS triton 0.1%) with 10% goat serum for 3 hours at RT. The samples were incubated for 3 days with the primary antibody (anti-GFP, living colors) at 4°C. Samples were washed in PBST for 3 hours and incubated in a biotinylated secondary antibody (Jackson ImmunoResearch) for 2 days at 4°C. Samples were finally washed for 3 additional hours at RT.
YFP signal was amplified with the Vectastain ABC Peroxidase kit and detected using the −3,3’-diaminobenzidine (DAB) Vectastain Elite ABC Kit (see the Key Resources Table). All procedures were performed as advised by the manufacturer. As a result, YFP⁺ cells and LacZ⁺ cells could be observed as brown and blue labels, respectively. Hearts were then stored in 80% glycerol at 4 °C.

Lioux G., Liu X., Temiño S., Oxendine M., Ayala E., Ortega S., Kelly R.G., Oliver G, & Torres M. (2020). A Second Heart Field-derived vasculogenic niche contributes to cardiac lymphatics. Developmental cell, 52(3), 350-363.e6.

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Immunohistochemical Analysis of Claudin-2, VDR, and TGR5

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Tissue sections from the TMA were deparaffinized, rehydrated through graded alcohols, and washed with phosphate-buffered saline. Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. The tissues were permeabilized with 0.3% Triton X for 1 h at room temperature. After endogenous peroxidase activity was quenched and nonspecific binding was blocked, mouse monoclonal anti-Claudin-2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-VDR (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-TGR5 antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated at 4 °C overnight. Biotinylated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was allowed to incubate for 1 h. After washing, sections were incubated with avidin-biotin–peroxidase complex (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. For color reaction development, slides were immersed in Vector NovaRed substrate (Vector Laboratories, Burlingame, CA) for 2 min and counterstained with Flex Hematoxylin for 2 min (Vector Laboratories, Burlingame, CA). A negative control was performed by replacing anti-VDR antibody with normal serum.

Abu-Farsakh S., Wu T., Lalonde A., Sun J, & Zhou Z. (2017). High expression of Claudin-2 in esophageal carcinoma and precancerous lesions is significantly associated with the bile salt receptors VDR and TGR5. BMC Gastroenterology, 17, 33.

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Quantifying Ischemic Lesion and Microglial Response

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All procedures have been described in detail previously [21 , 22 (link)]. Briefly, brains were perfusion fixed with 4% paraformaldehyde and cut into 40-μm thick coronal sections. Immunohistochemistry followed the peroxidase method with a biotinylated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania), ABC Elite reagent (Vector Laboratories, Burlingame, California), and diaminobenzidine (Sigma) as chromogen. The following primary antibodies were used: mouse anti-NeuN (MAB377B, Chemicon) 1:100 and rabbit anti-Iba1 (019-19741, Wako) 1:500. Lesion volume was quantified as described elsewhere [23 ]. The number of Iba1+ activated microglia per volume was assessed using StereoInvestigator® software (MicroBrightfield) as described previously [13 (link), 16 (link)]. Ischemic lesion size was measured by computer-assisted volumetry of serial 40-μm-thick NeuN-stained coronal brain sections (~ 2 mm apart) as described previously [24 (link)].

Boujon V., Uhlemann R., Wegner S., Wright M.B., Laufs U., Endres M., Kronenberg G, & Gertz K. (2019). Dual PPARα/γ agonist aleglitazar confers stroke protection in a model of mild focal brain ischemia in mice. Journal of Molecular Medicine (Berlin, Germany), 97(8), 1127-1138.

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Protein Expression and Antibody Staining

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Antibodies used were: anti-adenylyl cyclase 3 (AC3) (Santa Cruz Biotechnology, USA, #SC588), anti-olfactory marker protein (OMP) (Wako Chemicals, USA, #019-22291), anti-GAPDH (Millipore, USA, #MAB374), anti-FLAG (Sigma, USA, #F1804), peroxidase conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, USA, #31430), peroxidase conjugated anti-rabbit antibody (Thermo Fisher Scientific, #31460), and biotinylated secondary antibody (anti-mouse, rabbit and goat) (Jackson Immuno Research Labs, USA, #715-065-151, #711-065-152, and #705-065-003).
Reagents used were: Poly-D-lysine (Sigma, #P7886), Lipofectamine 2000 (Invitrogen, USA, #11668), puromycin (Sigma, #P3388), Normal Donkey Serum (Jackson Immuno Research Labs, #017-000-121), Protein A/G agarose beads (Calbiochem, USA, #IP05), FLAG magnetic M2 beads (Sigma, #M8823), Vectastain Elite ABC kit (Vector Laboratories, USA, #PK-6100), DAB peroxidase substrate kit (Vector Laboratories, #SK-4100), digitonin (Sigma, #D141), and CellLight ER-RFP, BacMam2.0 (Thermo Fisher Scientific, #C10591).

Ryu S.E., Shim T., Yi J.Y., Kim S.Y., Park S.H., Kim S.W., Ronnett G.V, & Moon C. (2017). Odorant Receptors Containing Conserved Amino Acid Sequences in Transmembrane Domain 7 Display Distinct Expression Patterns in Mammalian Tissues. Molecules and Cells, 40(12), 954-965.

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Immunohistochemical Detection of Polyglutamine

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For immunohistochemical detection, deparaffinized and rehydrated sections were used. Pretreatment in microwave 3 × 5 min at 800 W in the sodium citrate buffer (pH 6.0) and washing in 0.01 M PBS was mostly performed. For detection of polyglutamine deposits the pretreatment with 98% formic acid (5 min at room temperature) was required. Incubation in blocking solution (water solution of H₂O₂) for 20 min was followed by incubation with primary antibodies (Table 1) performed overnight at 4°C. Sections were then washed and incubated with the appropriate biotinylated secondary antibody (Jackson ImmunoResearch Lab., USA, 1 : 500) for 45 min at room temperature and subsequently with a streptavidin conjugate of peroxidase (Dako, CR) also for 45 min. Visualization of bound antibody was performed using DAB (Sigma-Aldrich, CR) and H₂O₂.

Mazurová Y., Anderova M., Němečková I, & Bezrouk A. (2014). Transgenic Rat Model of Huntington's Disease: A Histopathological Study and Correlations with Neurodegenerative Process in the Brain of HD Patients. BioMed Research International, 2014, 291531.

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Quantifying NKp46-HA Protein Binding

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The various recombinant HA proteins mentioned above were coated to an ELISA plate (0.1 μg/well). Blocking was performed with BSA 1%. 0.1 μg/well of NKp46-Ig or anti-HIS-Tag (clone AD1.1.10 from Bio-Rad, Kidlington, Oxford, England) were added followed by the relevant biotinylated secondary antibody from Jackson ImmunoResearch. The plate was then incubated with Streptavidin HRP (Jackson ImmunoResearch, West Grove, PA, USA). Binding was measured by using TMB solution and reading of the results was done with an ELISA reader at a wavelength of 650 nm. The binding of NKp46 to the different HA proteins was normalized to the amount of HA that was bound to the well, which we determined using the anti HIS-tag antibody.

Duev-Cohen A., Isaacson B., Berhani O., Charpak-Amikam Y., Friedman N., Drori Y., Mandelboim M, & Mandelboim O. (2020). Altered NKp46 Recognition and Elimination of Influenza B Viruses. Viruses, 13(1), 34.

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Immunohistochemical Staining Protocol

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For immunohistochemical staining of CD3, VCAM-1/ICAM-1, and B220, sections were incubated with the following primary antibodies: rat anti-human CD3 which reacts with mouse CD3 (Serotec, Raleigh, NC), rat anti-mouse VCAM-1 (Santa Cruz, Dallas, TX), rat anti-mouse ICAM-1 (Abcam, Cambridge, MA), and rat anti-mouse CD45R/B220 (BD Pharmingen, San Jose, CA), respectively, followed by a biotinylated secondary antibody (Jackson ImmunoResearch, West Grove, PA). For Ki-67 and Iba-1 staining, sections were individually incubated with rabbit anti-mouse Ki-67 (Vector Labs, Burlingame, CA) and rabbit anti-mouse Iba-1 (WAKO, Richmond, VA), followed by the DakoCytomation Envision+Dual Link System-HRP kit (Dako, Carpentaria, CA). For immunofluorescence staining, sections were incubated with rabbit anti-mouse glial fibrillary acidic protein (GFAP) (Millipore, Billerica, MA), followed by goat anti-rabbit IgG Alexa Fluor 594 (Jackson ImmunoResearch).

Wen J., Doerner J., Weidenheim K., Xia Y., Stock A., Michaelson J.S., Baruch K., Deczkowska A., Gulinello M., Schwartz M., Burkly L.C, & Putterman C. (2015). TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice. Journal of autoimmunity, 60, 40-50.

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