For immunofluorescence microscopy, 1.5 × 105 neutrophils were seeded onto coverslips coated with 0.001% poly-l -lysine (MilliporeSigma) and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Blocking was with 1% BSA overnight at 4°C. The primary antibody was against neutrophil elastase (Abcam catalog ab21595, diluted 1:100), and the FITC-conjugated secondary antibody was from Southern Biotech (catalog 4052-02, diluted 1:250). DNA was stained with Hoechst 33342 (Invitrogen). Images were collected with a Cytation 5 Cell Imaging Multi-Mode Reader.
Fitc conjugated secondary antibody
Manufactured by Southern Biotech
Sourced in United Kingdom
FITC-conjugated secondary antibodies are fluorescently labeled antibodies used to detect and visualize target proteins in various applications, such as immunohistochemistry, flow cytometry, and Western blotting. These antibodies bind to the primary antibodies that are specific to the target proteins, allowing for the detection and localization of the proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Easycyte flow cytometer, by Merck Group (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluoview 2000, by Olympus (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
Facsverse, by BD (1 mentions)
Aldehyde sulphate latex beads, by Thermo Fisher Scientific (1 mentions)
Anti cd9, by Abcam (1 mentions)
Perm wash buffer, by Southern Biotech (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Human umbilical vein endothelial cells (huvec), by ScienCell (1 mentions)
7 aad, by BD (1 mentions)
Enzyme free cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using fitc conjugated secondary antibody
1
Immunofluorescence Microscopy of Neutrophils
2
Neutrophil Elastase Immunofluorescence
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For immunofluorescence microscopy, 1.5 × 105 neutrophils were seeded onto coverslips coated with 0.001% poly-l -lysine (MilliporeSigma) and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Blocking was with 1% BSA overnight at 4°C. The primary antibody was against neutrophil elastase (Abcam, 21595, diluted 1:100), and the FITC-conjugated secondary antibody was from Southern Biotech (4052-02, diluted 1:250). DNA was stained with Hoechst 33342 (Invitrogen). Images were collected with a Cytation 5 Cell Imaging Multi-Mode Reader.
3
Quantification of Neutrophil Extracellular Traps
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NETs were detected by incubating with anti-human neutrophil elastase antibody (Abcam) for 1 h at 4°C, followed by incubation with FITC-conjugated secondary antibody (Southern Biotech) for an additional hour at 4°C. Nuclear DNA was stained with Hoechst 33342 (Invitrogen) for 10 min at room temperature. The chamber was gently removed from the attached slide, and ProLong Gold Antifade reagent (Invitrogen) was applied directly to the cells. The mount was secured in place by carefully lowering a coverslip onto the slide. Images were collected with a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). The percentage of NETs (decondensed extracellular DNA co-localized with neutrophil elastase) was determined as the average of six to eight fields (20x). Experiments were performed at least four times (independent biological replicates).
4
Evaluating FRα-Specific Antibody Binding to Native Protein
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Example 8
Flow cytometry studies were conducted to assess the ability of selected FRα-specific antibodies to bind to the native protein. For these studies Chinese hamster ovary (CHO) cells expressing FRα, were harvested, washed, and re-suspended in ice-cold growth media (RPMI supplemented with 10% FBS). Cells were incubated for 1 hour on ice with 9F3, 19D4, 24F12, or 26B3 (1 μg/mL), washed and then incubated with FITC-conjugated secondary antibodies [dilution 1:100] (Southern Biotech, Birmingham, Ala.). Prior to analysis, cells were labeled with 7-amino-actinomycin D (7-AAD) (BD Biosciences, Franklin Lakes, N.J.) for the exclusion of nonviable cells. CHO cells not expressing FRα were also subjected to the same experimental procedures, as a negative control. Cells were analyzed on an EASYCYTE™ Flow Cytometer (GUAVA® Technologies, Hayward, Calif.). The data provided in Table 4 indicate that all four antibodies are capable of binding native FRα.
5
Immunostaining for Stem Cell Markers
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Colonies grown at a density of 10 cells per well in 24 well plates for 2 weeks were fixed with 4% PFA for 20 min at room temperature. Colonies stained for intracellular markers (Ki67, K5, K18, BMI-1 and Oct4) were permeabilised by adding 200 µl 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 10 min at room temperature. Non-specific staining was blocked by incubating the colonies for 30 min with 10% normal goat serum (NGS) (PAA) at room temperature. Primary antibodies (Table S1 ) (Abcam, UK) diluted in 10% NGS in PBS were incubated overnight at 4°C, washed 4 times and incubated with FITC-conjugated secondary antibodies (Southern Biotech) for 1 h at room temperature. Finally, the colonies were washed, dried and mounted using Vectashield containing DAPI (Vector Laboratories Inc, Peterborough, U.K.). Colonies were viewed using Olympus Total Internal Reflection inverted confocal microscope and Fluoview 2000 software. The marker positive fraction was calculated as a percentage of total cell number (number of DAPI stained nuclei).
6
Characterization of Extracellular Vesicles via Flow Cytometry
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Fractions containing EVs were first identified according to their tetraspanin content determined by flow cytometry analysis. First, 50 µL of each fraction were incubated with 0.2 µL aldehyde/sulphate-latex beads (4 µm; Invitrogen, Carlsbad, CA) for 15 min at room temperature. Beads were then re-suspended in 1 mL bead-coupling buffer (BCB) (PBS supplemented with 0.1% BSA and 0.01% NaN3; Sigma–Aldrich) and incubated overnight at room temperature on rotation. EV-coated beads were then spun down at 2,000g for 10 min, washed with BCB and centrifuged again at 2,000g for 10 min. EV-coated beads were then labelled at 4°C with anti-CD9 (Clone VJ1/20), anti-CD63 (Clone TEA 3/18) (both kindly provided by Dr. Francisco Sánchez-Madrid and Dr. María Yañez-Mo), or polyclonal isotype (Abcam, Cambridge, UK) antibodies for 30 min. After washing with BCB, EV-coated beads were incubated with FITC-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL) for 30 min, washed twice with BCB and analysed by flow cytometry (FacsVerse; BD Biosciences, San Jose, CA) and using the Flow Jo software (Tree Star, Ashland, OR). A total of 10,000 beads/samples were acquired.
7
Intracellular Ref-1 Expression in Leukemia
8
Flow Cytometry Analysis of Cell Lines
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Aortic Smooth Muscle Cells (Invitrogen), HUVEC (ScienCell, Carlsbad, CA) cells, and SJSA-1 osteosarcoma cells (American Type Culture Collection (ATCC), Manassas, VA) were harvested using enzyme-free cell dissociation buffer (Invitrogen) or trypsin as indicated. Cells were washed twice in PBS and re-suspended at 5X104 cells/100 uL FACS buffer (PBS+2% FBS) in a round bottom 96 well plate. Cells were incubated for 1hr on ice with primary antibodies at a concentration of 10 μg/mL, washed and then incubated with FITC-conjugated secondary antibodies (dilution 1:100) (Southern Biotech). Prior to analysis, cells were labeled with 7-AAD (BD Biosciences, Franklin Lakes, NJ) for the exclusion of nonviable cells. Cells were analyzed on an EasyCyte Flow Cytometer (Guava Technologies, Hayward, CA).
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