Cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 20 min. After washed by PBS three times, cells were blocked with 5% BSA for 30 min and then incubated with biotinylated-MAL II (Vector Laboratories) overnight at 4 °C. Cells were incubated with FITC-Avidin (#405101; BioLegend, USA) for 1 h. The cells were washed by PBST and counterstained with 2 μg/mL Hoechst (#33342; Thermo Scientific) for 5 min. Images were obtained using a fluorescence microscope (DM6 B; Leica, Germany).
Fitc avidin
Manufactured by BioLegend
Sourced in United States
FITC-avidin is a fluorescent conjugate of the protein avidin. Avidin is a glycoprotein that binds to the small molecule biotin with high affinity. FITC-avidin can be used to detect and localize biotinylated proteins, nucleic acids, and other biomolecules in various applications such as flow cytometry, fluorescence microscopy, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst, by Thermo Fisher Scientific (1 mentions)
Biotinylated malii, by Vector Laboratories (1 mentions)
Biotin conjugated dba lectin, by Merck Group (1 mentions)
Accuri c6, by BD (1 mentions)
Fetal bovine serum (fbs), by Zhejiang Tianhang Biotechnology (1 mentions)
1640 medium, by Zhejiang Tianhang Biotechnology (1 mentions)
Alexa fluor 594 goat anti rat igg, by Abcam (1 mentions)
Bz x800 fluorescence microscope, by Keyence (1 mentions)
Fluoromount g containing dapi, by Thermo Fisher Scientific (1 mentions)
Biotin fitc, by Merck Group (1 mentions)
Anti his tag, by Cell Signaling Technology (1 mentions)
Anti mouse f4 80 antibody, by BioLegend (1 mentions)
Plga lactide glycolide 50 50, by Merck Group (1 mentions)
Cd206, by BD (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n, by Avanti Polar Lipids (1 mentions)
Tnf α, by BD (1 mentions)
5 protocols using fitc avidin
1
Lectin Staining for Glycoprotein Imaging
2
Isolation and Characterization of Endometrial Cells
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Cell suspensions were generated by shredding and grinding the endometrial tissues in 3 ml of 1640 medium supplemented with 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd, China). After filtration and centrifugation, a mixture of 4 ml of cell suspension and 4 ml of lymphocytes separation medium (Tianjin Haoyang Biological Manufacture Co., Ltd, China) was centrifuged at 1350 g for 30 min at 20°C. After washing the samples twice, the cell suspension was incubated with anti-CD32 (0.1 μl/106 cells in 100 μl buffer, BD Biosciences, USA) for 10 min at 4°C to block Fc receptors (FcRs). Samples were then incubated with biotin-conjugated DBA-lectin (1 μl/106 cells in 100 μl buffer, Sigma-Aldrich, USA) for 3 min, and the suspension was incubated with FITC-avidin (0.25 μl/106 cells in 100 μl buffer, BioLegend Inc., USA) for 30 min at 4°C. After the incubation period, samples were analyzed using a flow cytometer (Accuri C6, BD Biosciences, USA).
3
Quantifying Mast Cells in Mouse Skin
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The mouse ears were fixed in 10% formamide and embedded in paraffin. Sections (2 μm) were deparaffinized and autoclaved at 121°C for 10 min in 10 mM sodium citrate buffer (pH 6.0). The sections were blocked with 5% skimmed milk in TBS-T at room temperature for 60 min and then incubated with rabbit anti-mMCP-6 antibody (R&D Systems, MN, USA, 1:500) at 4°C overnight. After washing with TBS, sections were incubated with secondary antibody (Alexa Fluor 594 goat anti-rat IgG, Abcam, 1:500) and FITC-avidin (BioLegend, CA, USA, 1:200) for 1 h at room temperature and counterstained with DAPI. FITC-avidin was used to label mast cells in the skin. Images were captured using a BZ-X800 fluorescence microscope (KEYENCE, Osaka, Japan). The digitized images were transferred to a computer to measure the size of each region using a software (ImageJ, National Institute of Mental Health, MD, USA), and FITC-positive mast cells were counted. The results are expressed as positive cells per mm2.
4
Immunofluorescence Staining of Paraffin Sections
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Paraffin sections (5 µm) were deparaffinized as follows: 2 x 10 min in xylol, 2 x 3 min in absolute ethanol, 2 x 3 min in 96 % ethanol, 1 x 3 min in 70 % ethanol, 3 x 5 min in deionized water and 3 x 5 s in TBS buffer (Tris-Base (7,4 mmol/L), Tris-HCl (43,5 mmol/L), NaCl (150 mmol/L), pH = 7.5). For MC staining, the sections were incubated with Avidin-FITC (BioLegend) for 15 min in the dark. After washing (3 x 3 min with TBS) the sections were embedded with Fluoromount-G™ containing DAPI (Thermo Fisher Scientific) and dried for 24 h.
5
Characterization of Nanoparticle-Based Therapies
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SnMP, DNR hydrochloride, poly(lactic‐co‐glycolic acid) (PLGA, lactide: glycolide 50:50, 7000–17000 Da), and biotin‐FITC were purchased from Sigma Aldrich (St. Louis, MO, USA). 1,2‐Dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[biotinyl(polyethylene glycol)‐2000] (DSPE‐PEG2000‐Biotin), 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[amino(polyethylene glycol)‐2000] (DSPE‐PEG2000) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Anti‐human CD33, CD64 antibodies and anti‐mouse CD11b, CD45, CD206, Ly6c, Gr1, TNF‐α, IL12p70, Rat IgG1 Isotype antibodies were purchased from BD Biosciences (USA) (Table 1, Supporting Information). Anti‐mouse F4/80 antibody and Avidin‐FITC were purchased from Biolegend (San Diego, CA, USA). Anti‐human CCR2 antibody was obtained from R&D Systems (Minneapolis, MN, USA). Anti‐His‐Tag, anti‐human HO1 (P249), and β‐actin antibodies (13E5) were obtained from Cell Signaling Technology (Danvers MA, USA).
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