Appropriate biotinylated secondary antibodies

Manufactured by Vector Laboratories

Appropriate biotinylated secondary antibodies are laboratory reagents used to detect and visualize target proteins in various biological assays. These antibodies are conjugated with biotin, a small molecule that can bind strongly to the protein avidin or streptavidin. This biotinylation allows for signal amplification and enhanced detection sensitivity.

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Biotinylated antibodies (8 citations) Appropriate secondary antibodies (3 citations)

7 protocols using appropriate biotinylated secondary antibodies

Multidisciplinary Immunohistochemical Analysis

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All spinal cord and optic nerve fixed tissues as well as human brain tissue, lymph nodes, normal sural nerve, cutaneous neurofibromas or plexiform neurofibromas, and mouse sciatic nerve, cutaneous or plexiform neurofibromas were paraffin-embedded, serially sectioned (5 μm) and immunostained with GFAP, Iba1, Ki67, CD3, Midkine, GAP43, CD34, Factor XIIIa, SOX10, neurofilament-200, and Col1a2 (Supplementary Table 1). Immunohistochemical staining was performed using the Vectastain ABC kit (Vector Laboratories) and appropriate biotinylated secondary antibodies (Vector Laboratories). Hematoxylin and eosin (H&E) staining was performed following the manufacturer’s instructions (StatLab). Primary RGCs, hippocampal neurons, DRG neurons, shNF1 Schwann cells, and hiPSC-sensory neurons were immunostained with appropriate primary (RGCs: Rbpms, Tuj-1; hippocampal neurons: GAD65, Glutamine synthetase, Tuj-1; DRG neurons: Peripherin, ISL1, Tuj-1; SCs: EGR2, S100β, OCT6, SOX10; sensory neurons: peripherin, BRN3A, SMI32, ISL1, p75NTR, Nestin, Tuj-1) and secondary Alexa-fluor-conjugated antibodies (Supplementary Table 1). Images were acquired using Image Studio Lite Version 5.2 software, and LAS AF Lite 3.2.0 software and analyzed using ImageJ 1.53a software, as well as Adobe Photoshop version 21.1.1.

Anastasaki C., Mo J., Chen J.K., Chatterjee J., Pan Y., Scheaffer S.M., Cobb O., Monje M., Le L.Q, & Gutmann D.H. (2022). Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1. Nature Communications, 13, 2785.

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Immunohistochemical Analysis of Bone Markers

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Rehydrated slides were blocked with 10% goat serum; the primary antibodies used included rabbit polyclonal anti-pSMAD1/5/8, anti-OCN, anti-RUNX2, anti-SOX9, and anti–collagen-2 in 1% rabbit serum (1:80 dilution, Santa Cruz Biotechnology). The appropriate biotinylated secondary antibodies were used in 1:1000 dilution (Vector Laboratories). Visualization was performed with diaminobenzidine (Zymed Laboratories), as previously described (33 (link)).

Peterson J.R., De La Rosa S., Eboda O., Cilwa K.E., Agarwal S., Buchman S.R., Cederna P.S., Xi C., Morris M.D., Herndon D.N., Xiao W., Tompkins R.G., Krebsbach P.H., Wang S.C, & Levi B. (2014). Treatment of heterotopic ossification through remote ATP hydrolysis. Science translational medicine, 6(255), 255ra132.

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Immunohistochemical Analysis of CD68 and α-SMA

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Slides were deparaffinized with xylene and rehydrated with a graded ethanol series. Antigen retrieval was performed, and specimens were blocked prior to incubation with primary antibodies. Primary antibodies included CD68 (1:250 dilution ratio; Abcam, Cambridge, Massachusetts) and α-SMA (α-smooth muscle actin, 1:2000 dilution ratio; Dako, Carpinteria, California). Antibody binding was detected with appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame, California), followed by binding of streptavidin-horseradish peroxidase (Vector Laboratories) and color development with 3,3-diaminobenzidine (Vector Laboratories).

Pepper V., Best C.A., Buckley K., Schwartz C., Onwuka E., King N., White A., Dharmadhikari S., Reynolds S.D., Johnson J., Grischkan J., Breuer C.K, & Chiang T. (2019). Factors Influencing Poor Outcomes in Synthetic Tissue-Engineered Tracheal Replacement. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery, 161(3), 458-467.

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Immunostaining Protocol for Antigen Detection

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A general procedure is described for immunostaining. In brief, antigen retrieval was performed by incubating slides with Epitope Retrieval Solution (IHCWORD) at 95°C for 20 minutes, or Ficin digestion for 10 minutes at room temperature. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide. Slides were blocked with 5% goat serum and incubated with primary antibodies. Antibodies include rabbit polyclonal anti-GFP (Cell Signaling Technology), and Ki67 (Thermo scientific). Appropriate biotinylated secondary antibodies were used (Vector Laboratories) and signal is either detected using DAB (Vector Laboratories) with the addition of NiCo or Alexa 488 conjugated secondary antibody (Invitrogen) followed by DAPI mounting medium (Vector Laboratories). TUNEL staining was performed as per the manufacturer's instructions (Roche).

Dhamdhere G.R., Fang M.Y., Jiang J., Lee K., Cheng D., Olveda R.C., Liu B., Mulligan K.A., Carlson J.C., Ransom R.C., Weis W.I, & Helms J.A. (2014). Drugging a Stem Cell Compartment Using Wnt3a Protein as a Therapeutic. PLoS ONE, 9(1), e83650.

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Immunohistochemical Staining of Progranulin

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Hydrogen peroxide (3%) in 60% methanol was used to eliminate endogenous peroxidase activity. These sections were blocked with serum (Jackson ImmunoResearch) in 0.2% Triton-X in 1X PBS for 1 h at 37°C. Sections were then incubated overnight at 4°C with anti-progranulin antibody (R & D Systems; AF2420; 1:400) or anti-IBA1 (Wako; 019-19741; 1:500). Appropriate biotinylated secondary antibodies (Vector Labs) were then applied at 1:500 and incubated for 1 h at 37°C. Staining was performed using the avidin-biotin (Vector Labs) complex method, and tissue developed for 15 min using 3,3′-diaminobenzidine tablets (Sigma-Aldrich).

Sarkar S., Dammer E.B., Malovic E., Olsen A.L., Raza S.A., Gao T., Xiao H., Oliver D.L., Duong D., Joers V., Seyfried N., Huang M., Kukar T., Tansey M.G., Kanthasamy A.G, & Rangaraju S. (2020). Molecular Signatures of Neuroinflammation Induced by αSynuclein Aggregates in Microglial Cells. Frontiers in Immunology, 11, 33.

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Immunohistochemical Staining of Brain Tissue

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Paraffin embedded brain sections were subjected to IHC staining. Antigen retrieval of brain slides was performed by microwave for 10 min in a chamber containing citrate buffer (0.01 M; pH 6.0). Following antigen retrieval, tissue slides were placed in 0.3% methanol/hydrogen peroxidase (Sigma-Aldrich Co.) for 30 min to quench the endogenous peroxidase. To block the non-specific binding of antibodies, this slide was incubated with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h. Subsequently, the slides were incubated overnight with a mixture of anti-PCNA (1:100, Abcam plc., Cambridge, UK) primary antibody in 5% bovine serum albumin (Sigma-Aldrich Co.) in a humidified chamber. The next day, the slides were washed three times in 1 × PBS-T (pH 7.4) and then incubated with appropriate biotinylated secondary antibodies (Vector Laboratories) for 30 min at room temperature. Slides were subsequently rinsed with PBS-T for 10 min, after which Vectastain Universal Elite ABC kit reagent (Vector Laboratories) was applied for 30 min. Immunoreactive complexes were detected using DAB substrate (Sigma-Aldrich Co.) and counted after staining with hematoxylin. Finally, slides were mounted with a cover slip using mounting medium. All slides were visualized under a BX51 light microscope for digital photography.

Yi B.R., Kim S.U, & Choi K.C. (2015). Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer. Oncotarget, 7(5), 5985-5999.

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Immunohistochemical Analysis of Liver Samples

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Liver or tumor samples were fixed in 10% neutral buffered formalin (Fisher Scientific, Grand Island, NY, USA), embedded in paraffin, and sectioned to 10 μm thickness. The sections were de-paraffinized by xylene and then incubated in antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) for 10 min at 100°C, and followed by incubation with 3% hydrogen peroxide solution in methanol to quench endogenous peroxidase activity in sections. The sections were then blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories) and incubated with the antibody against anti-p62 (Novus Biologicals, NBP1–49956) or anti-β-catenin (Cell Signaling, #9582) and followed with the appropriate biotinylated secondary antibodies (Vector Laboratories). The resulting complexes in the sections were visualized with an ABC kit using the DAB Substrate (Vector Laboratories). The sections were also counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) and visualized using a Zeiss Axioskop2 plus microscope equipped with 10X/0.45NA, 20X/0.5NA or 40X/0.75NA objectives (Carl Zeiss, Oberkochen, Germany).

Cho J.H., Lee Y.M., Bae S.H, & Chou J.Y. (2019). Activation of tumor-promoting pathways implicated in hepatocellular adenoma/carcinoma, a long-term complication of glycogen storage disease type Ia. Biochemical and biophysical research communications, 522(1), 1-7.

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