Streptavidin fitc

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany

Streptavidin-FITC is a conjugate of the protein streptavidin and the fluorescent dye fluorescein isothiocyanate (FITC). Streptavidin has a high affinity for the vitamin biotin, making it a useful tool in various biotechnological applications.

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Close spelling variants of this product

Streptavidin fluorescein isothiocyanate (FITC) conjugate (2 citations) FITC-conjugated streptavidin (24 citations) FITC-labeled streptavidin (2 citations) Streptavidin FITC conjugate (1 citations) Biotin-conjugated anti-rabbit IgG and FITC-labeled streptavidin (2 citations)

60 protocols using streptavidin fitc

Immunohistochemical Evaluation of Misfolded SOD1

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Tissues were de-paraffinized through histology grade CitriSolv (three times for 5 minutes each) and a graded alcohol series (100%, 95% and 70% ethanol (vol/vol) twice for 5 minutes each). After a 10 minute permeabilization step in 1x PBS, 0.2% TritonX100, antigen retrieval (10mM Citrate buffer, pH6.0, in pressure cooker at 120°C for 20 minutes) was applied to the sections. This antigen retrieval step was skipped when the effects of the absence of antigen retrieval on the immunostaining were being tested. Sections were further blocked with 10% normal goat serum (vol/vol, Jackson ImmunoResearch Laboratories) and incubated with misfolded SOD1 antibodies (C4F6 (1/200) and B8H10 (1/1,000; MediMabs) and 3H1 (1:10,000; gift from Dr. Cashman) with or without rabbit TDP-43 (1/50; Proteintech #10782-2) prepared in antibody diluent (Dako) overnight at 4°C. Sections were then incubated with biotinylated secondary antibodies (Jackson ImmunoResearch Laboratories) followed by streptavidin-FITC (Invitrogen). To reduce autofluorescence noise, quenching with 0.1% Sudan Black in 70% EtOH for 5–10 seconds was applied prior to coverslip mounting with Prolong (Invitrogen). Imaging was performed on a Nikon Eclipse laser scanning confocal microscope.

Da Cruz S., Bui A., Saberi S., Lee S.K., Stauffer J., McAlonis-Downes M., Schulte D., Pizzo D.P., Parone P.A., Cleveland D.W, & Ravits J. (2017). Misfolded SOD1 is not a primary component of sporadic ALS. Acta neuropathologica, 134(1), 97-111.

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Immunofluorescence Staining of PSA

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A solution of 4% paraformaldehyde was used to fix the cells for 30 min prior to block the non-specific binding with 0.5% BSA in PBS for 5 min. Cells were then incubated with 0.01 mg/ml of biotinylated PSA (Vector Laboratories) at 4°C, overnight and 1 h at room temperature with 1:500 streptavidin-FITC (Invitrogen, UK). Hoechst 33342 (Molecular probe, Invitrogen) was used to stain the nuclei and the fluorescent image was visualized using a ZEISS LSM 800 Confocal Laser Scanning Microscope (Zeiss, Oberkochen, Germany). The intensities of the fluorescent signals were analyzed using ZEN 2.1 software (Zeiss).

Phoomak C., Silsirivanit A., Park D., Sawanyawisuth K., Vaeteewoottacharn K., Wongkham C., Lam E.W., Pairojkul C., Lebrilla C.B, & Wongkham S. (2018). O-GlcNAcylation mediates metastasis of cholangiocarcinoma through FOXO3 and MAN1A1. Oncogene, 37(42), 5648-5665.

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Quantifying Sporozoite Gliding Motility

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To quantify sporozoite gliding motility, a glass eight-well chamber slide was coated with 10 μg/ml anti-CSP antibody in 1× PBS overnight, and the assay was performed as described previously (Stewart & Vanderberg, 1988 (link)). SG sporozoites in 3% BSA/DMEM were added 5,000/well and incubated for 1 h at 37°C. After fixation with 4% PFA, trails were visualized by staining with biotinylated anti-CSP antibody, followed by streptavidin-FITC (19538-050; Invitrogen). Trails associated with sporozoites were counted using a Nikon Eclipse 80i fluorescent microscope using a Nikon Plan Fluor 40×/0.75 objective. Pictures of trails associated with sporozoites were acquired using Leica LAS-X software on a confocal laser scanning microscope (Leica TCS SP-8) with an HC PL APO 40×/0.85 objective.

Choudhary H.H., Gupta R, & Mishra S. (2019). PKAc is not required for the preerythrocytic stages of Plasmodium berghei. Life Science Alliance, 2(3), e201900352.

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Quantifying Stem Cell Populations

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On the tenth day of treatment with TGF-β1 and/or asporin peptide, the cells were harvested with trypsin, and 2.5 × 10⁵ cells were suspended in 25 μl of PBS and labeled with 1/100 (0.01 mg/ml) anti-CD24 (biotinylated; BioLegend, catalog no. 101803) for 1 h at 4°C. Following a wash, the cells were further labeled with 1/100 (0.01 mg/ml) anti-CD44 (PE-labeled; eBioscience, catalog no. 12–0441) and 1/1,000 streptavidin-FITC (Invitrogen, catalog no. SA100-02) for 1 h at 4°C. Following this, the cells were washed two times, and 1/50 7-aminoactinomycin D (7-AAD; BD-Pharmingen, catalog no. 51-68981E) was added for 10 min. The cell suspensions were analyzed using a FACSAria flow cytometer (BD Biosciences). Stem cells were quantified by evaluating the percentage of 7-AAD^neg, CD44^high/CD24^low cell population.

Maris P., Blomme A., Palacios A.P., Costanza B., Bellahcène A., Bianchi E., Gofflot S., Drion P., Trombino G.E., Di Valentin E., Cusumano P.G., Maweja S., Jerusalem G., Delvenne P., Lifrange E., Castronovo V, & Turtoi A. (2015). Asporin Is a Fibroblast-Derived TGF-β1 Inhibitor and a Tumor Suppressor Associated with Good Prognosis in Breast Cancer. PLoS Medicine, 12(9), e1001871.

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Isolation and Characterization of Malaria Antigen-Specific Memory B Cells

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Frozen PBMCs were thawed, washed with RPMI 1640 (Gibco) and incubated with full‐length biotinylated NF54 PfCSP in the presence of the following mouse anti‐human antibodies at the indicated dilutions: CD27‐phycoerythrin (PE) (Clone ID: M‐T271; Cat. No: 555441) at 1:5, CD38‐ Brilliant Violet 605 (BV605) (Clone ID: HB7; Cat. No: 562665) at 1:20, IgG‐BV510 (Clone ID: G18‐145; Cat. No: 563247) at 1:20, IgM‐BV421 (Clone ID: G20‐127; Cat. No: 562618) at 1:20, IgD‐Allophycocyanin‐H7 (APC‐H7) (Clone ID: IA6‐2; Cat. No: 561305) at 1:20 (all from BD Biosciences), CD19‐BV786 (Clone ID: HIB19; Cat. No: 302240) at 1:10 and CD21‐ PE‐Cy7 (Clone ID: Bu32; Cat. No: 354912) at 1:20 (both from BioLegend). Biotin was detected using streptavidin FITC (Invitrogen; Cat. No: 11‐4317‐87) at 1:1,000 dilution. 7‐Aminoactinomycin D (7‐AAD) (Invitrogen; Cat No: A1310) at 1:400 was used as a marker for dead cells. Single‐cell sorting of 7AAD−PfCSP+CD19+ B cells gated positive for either IgG or CD27 as PfCSP memory B cells and of 7AAD−CD38+CD27+CD19+ cells as plasmablasts was performed using a FACS Aria II (BD Biosciences) with FACSDiva software (version 8.0.1) using the index sort option. Data were analyzed using FlowJo v.10.0.8 (Tree Star).

Oludada O.E., Costa G., Burn Aschner C., Obraztsova A.S., Prieto K., Canetta C., Hoffman S.L., Kremsner P.G., Mordmüller B., Murugan R., Julien J., Levashina E.A, & Wardemann H. (2023). Molecular and functional properties of human Plasmodium falciparum CSP C‐terminus antibodies. EMBO Molecular Medicine, 15(6), e17454.

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Immunohistochemical Analysis of Murine Thymus

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Adult (2 month-old) and neonatal (day 9) thymi were isolated, embedded in Tissue-Tek O.C.T compound (Sakura Finetek, U.S.A.) and snap frozen in a liquid nitrogen/isopentane slurry. Sections of 5-8μm were cut using a Microm HM550 Cryostat (Thermo Scientific). Sections were air-dried for 20 min and stored at -80°C prior to staining. Sections were blocked with 5% (v/v) goat serum in PBS with 0.5% Tween-20 (v/v) for 30 min at room temperature before incubation with primary antibodies for 30 minutes. Primary antibodies included biotinylated anti-mouse pan-keratin (LifeSpan BioSciences, clone Lu-5), anti-mouse K5 (Covance, clone Poly 19055), biotinylated UEA-1 lectin (Vector labs, USA), anti-mouse DEC205-FITC (clone NLDC145) and anti-mouse AIRE-Alexa647 (clone 5H12), anti-mouse K8 (clone Troma-I, DSHB) and ER-TR7 (provided by Prof Richard Boyd, Monash University). Following three washes in PBS, 5 min each, sections were then incubated with appropriate secondary antibody (anti-rabbit Ig Alexa-555 (Life Technologies)) and streptavidin FITC (Invitrogen) for 30 min, counterstained with DAPI (Sigma-Aldrich), then mounted with Vectashield (Vector labs). Images were collected using a LSM780 confocal with Zen 2012 SP2 (black) software v11.0 (Zeiss). Single optical sections and maximal intensity projection images were processed for presentation using OMERO29 (link).

Jain R., Sheridan J.M., Policheni A., Heinlein M., Gandolfo L.C., Dewson G., Smyth G.K., Sansom S.N., Fu N.Y., Visvader J.E., Holländer G.A., Strasser A, & Gray D.H. (2017). A Critical Epithelial Survival Axis Regulated by MCL-1 Maintains Thymic Function in Mice. Blood, 130(23), 2504-2515.

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Immunostaining of Cells with PSA

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A solution of 4% paraformaldehyde was used to fix the cells for 30 min prior to block the non-specific binding with 0.5% BSA in PBS for 5 min. Cells were then incubated with 0.01 mg/ml of biotinylated PSA (Vector Laboratories) at 4 °C, overnight and 1 h at room temperature with 1:500 streptavidin-FITC (Invitrogen, UK). Hoechst 33342 (Molecular probe, Invitrogen) was used to stain the nuclei and the fluorescent image was visualized using a ZEISS LSM 800 Confocal Laser Scanning Microscope (Zeiss, Oberkochen, Germany). The intensities of the fluorescent signals were analyzed using ZEN 2.1 software (Zeiss).

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Lipid Labeling and Microsphere Functionalization

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All lipids in this study were purchased from Avanti Polar Lipids, Inc. DiO (DiOC₁₈(3) (3,3′-dioctadecyloxacarbocyanine perchlorate)), DiD (DiIC18(5) ((1,1′-Dioctadecyl-3,3,3′,3′-Tetra-methylindodicarbocyanine Perchlorate)), streptavidin-FITC, and Alexa Fluor633-strepavidin were obtained from Invitrogen (Molecular Probes). Silica microspheres of 5 μm nominal size were obtained from Bangs Laboratories, Inc. Anti-FLAG-FITC was obtained from Sigma-Aldrich. The AlphaLISA reagents were purchased from PerkinElmer Inc. (Waltham, MA).

Gilchrist M.L., Ahn K, & Li Y.M. (2016). Imaging and Functional Analysis of γ-Secretase and Substrate in a Proteolipobead System with an Activity-Based Probe. Analytical chemistry, 88(2), 1303-1311.

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Tumor Vasculature Imaging Protocol

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Cryosections of tumors were stained with anti-CD31 (eBioscience) and anti-phospho-Stat 3 (Santa Cruz) antibodies, respectively, followed by a biotinylated secondary antibody (Santa Cruz) and streptavidin-FITC (fluorescein isothiocyanate) with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) counterstaining to detect tumor vasculature. The fluorescence images were taken by a microscope (NIKON ECLIPSE TI-SR, Japan), and processed by using Image pro-plus 6.0 software.

Xu H., Zou S., Xu X, & Zhang L. (2016). Anti-tumor effect of β-glucan from Lentinus edodes and the underlying mechanism. Scientific Reports, 6, 28802.

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Multiparametric Flow Cytometry Analysis

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Cells were stained with a combination of surface marker antibodies detailed below. Primary direct staining was performed with antibodies purchased from Biolegend: FITC anti-CD86, clone RMMP-2; FITC anti-MHC-II (I-A/I-E), clone M5/114.15.2; FITC anti-CD25, clone 3C7; PE anti-MHC-I, clone AF6-88.5; PE Biotin anti-CD80, clone 1610A1; PE anti-PDL1, clone 10F.9G2; PE anti-CD137, clone 17B5; APC/Cy7 anti-CD69, clone H1.2F3. For indirect staining antibodies were purchased from Biolegend: Biotin anti-IFN-γ, clone XMG1.2; from eBioscience: Biotin anti-SIINFEKL/H2-K^b, clone 25-D1.16. Here, applicable cells were stained with secondary Streptavidin-FITC (Invitrogen) or FITC anti-rat IgG, clone Poly4054 (Biolegend). Staining was carried out for 20–30 min at 4°C in FACS buffer containing 0.5% sodium azide in PBS. Samples were acquired using the Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, USA) and analyzed using FlowJo software. Fold change in MFI was calculated by using the following formula: fold change = [(SIINFEKL MFI − Negative MFI)/Negative MFI], where “SIINFEKL” refers to cells that were pulsed with SIINFEKL peptide and “Negative” refers to unpulsed cells.

Mulder R., Banete A., Seaver K, & Basta S. (2017). M(IL-4) Tissue Macrophages Support Efficient Interferon-Gamma Production in Antigen-Specific CD8+ T Cells with Reduced Proliferative Capacity. Frontiers in Immunology, 8, 1629.

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