Complement deposition was analyzed by flow cytometry. S. pyogenes was grown to mid-exponential phase (OD600nm ~ 0.5). After a wash with PBS, 300 µl bacterial suspensions (5x106 CFU) in HEPES buffered saline (HBS++: 20 mM HEPES, 140 mM NaCl, pH 7.4, 2.5 mM MgCl2, 5 mM CaCl2) containing 0.01% BSA were mixed with 300 µl freshly prepared human serum containing rCEF for 50 min at 37°C with constant shaking. The mixture was then washed twice with 1.5 ml PBS supplemented with 1% BSA. The pellet was resuspended in 300 µl of PBS-1% BSA containing 5 µg/ml of either rabbit anti-C5b-9 or rabbit anti-C3b antibodies (Abcam) and incubated at RT for 15 min. Subsequent to the wash step, the bacteria were resuspended in 300 µl of FITC-labeled secondary antibody (Abcam) at a ratio of 1:100 in PBS-1% BSA and incubated for 15 min at RT. Finally, the bacteria were washed and resuspended in 800 µl of PBS for flow cytometry analysis using a BD LSRII flow cytometer (BD Biosciences). An opsonised sample containing bacteria and serum and a non-opsonised sample (bacteria only) were used as controls.
Fitc labeled secondary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
FITC-labeled secondary antibody is a fluorescently-conjugated antibody that binds to the primary antibody, allowing for visualization and detection of target proteins in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometry, by Beckman Coulter (1 mentions)
Bca kit, by Keygen Biotech (1 mentions)
Eclipse 50i fluorescence microscope, by Nikon (1 mentions)
Anti insulin primary antibody, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Infinite m200, by Tecan (1 mentions)
Tcs sp5 confocal fluorescence microscope, by Leica (1 mentions)
Anti sox9, by Abcam (1 mentions)
Vectashield, by Zeiss (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Anti ve cadherin antibody, by Abcam (1 mentions)
Ckx53 microscope, by Olympus (1 mentions)
Ve cadherin, by Abcam (1 mentions)
Fluorescent microscopy, by Olympus (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Collagen 3, by Abcam (1 mentions)
16 protocols using fitc labeled secondary antibody
1
Analysis of Complement Deposition on Streptococcus pyogenes
2
Bronchoalveolar Lavage Neutrophil Quantification
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The left lung was lavaged three times with 0.8 ml PBS. The collected lavage fluid was subjected to centrifugation for 10 min at 1000 g and 4 °C. The supernatant was collected for use as BALF, and the total protein content was measured using a BCA kit (KeyGen Biotech, Nanjing, China). To calculate the percentage of neutrophils in BALF, the collected BALF cells were resuspended in PBS supplemented with 10% fetal calf serum (Gibco, Grand Island, NY, USA). A sample of 5 × 105 cells was probed by a specific anti-neutrophil antibody (Abcam, Cambridge, MA, USA) for 30 min and then incubated with a FITC-labeled secondary antibody (Abcam) for another 30 min. The fluorescence intensity of each sample was analyzed by CytoFLEX flow cytometry (Beckman, CA, USA).
3
Immunofluorescent Labeling of Islet Grafts
4
Immunofluorescence Analysis of Osteoblast Markers
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For the immunofluorescence analysis, MC3T3-E1 cells were cultured on various microcarriers for 7 days at 37.8 °C in a humidified 5% CO2 atmosphere. The samples were permeated with 0.1% Triton-X 100 in phosphate buffer for 5 min. After being blocked using 1% BSA in phosphate buffer for 30 min, the samples were incubated with primary antibody (OPN and Runx2, 1:500, Abcam) for 60 min under ambient temperature. Then, cell/microcarriers samples were washed with PBS for three times and stained with a fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:500, Abcam) for 60 min. Finally, cell nuclei were dyed with 4′,6-diamidino-2-phenylindole (DAPI). Photos were taken on a multifunctional microplate scanner (Tecan Infinite M200).
5
NF-κB p65 Immunofluorescence Imaging
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FLS cells were fixed in 4% methanol at 4°C for 15 min, washed three times (5 min each) with PBS, permeabilized with 0.1% Triton X-100 for 15 min, then, incubated overnight at 4°C with anti-NF-κB p65 primary antibody (1:100; cat. no. ab32536; Abcam), followed by incubation with FITC-labeled secondary antibody (cat. no. ab7086; Abcam) at room temperature for 1 h. Then, the cells were counterstained with DAPI for 5 min. Images were captured using a Leica TCS SP5 confocal fluorescence microscope (magnification, ×80). The negative control was prepared in the same manner without the primary antibody.
6
Characterize Rat Neural Progenitor Cells
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According to the prior studies, three major specific cell surface markers of rat NPCs (N-CDH, KRT19, and SOX9) were identified by the fluorescence activated cell sorter (FACS) using flow cytometry 20 (link)-22 (link). Briefly, the isolated cells were trypsinized, centrifuged and resuspended in PBS. Then the cells were fixed and permeabilized by fixation and permeabilization kit, and incubated successively with primary antibodies (anti-N-cadherin (N-CDH, Proteintech, China), anti-keratin19 (KRT19, Proteintech, China), and anti-SOX9 (Abcam, USA)), and FITC-labeled secondary antibody (Abcam, USA). Finally, the expression of fluorescent intensity was quantitatively determined by the flow cytometry.
7
Visualizing Cellular Uptake of Diverse DNA Sources
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NIH3T3 mouse fibroblast cells grown in 35 mm3 dishes on cover slips (10 × 104 cells) were treated with fluorescently-labeled HMW and sDNA (10 ng each) isolated from WI-38, MDA-MB-231, bacterial and plant sources. After 30 min of treatment, cells were washed ×3 with PBS and, with exception of cells treated with plant DNA (see below), were processed for LSCM using anti-BrdU antibody (1:100 dilution) (Abcam, USA, Catalogue No. ab6326) and FITC-labeled secondary antibody (1:500 dilution) (Abcam, USA, Catalogue No. ab102263), mounted onto clean glass slides with Vecta-shield and were visualized using Zeiss differential LSCM platform. Since plant DNA was non-enzymatically labeled in vitro, the treated cells were directly visualized under LSCM platform. Fifty nuclei were randomly chosen for analysis in each case and the mean nuclear fluorescence intensity was measured using LSM Image Examiner 4.0 software (Carl Zeiss Jena GmbH, Germany).
8
Isolation and Characterization of Cardiac Fibroblasts
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Third-generation cardiac fibroblasts were prepared into a single cell suspension with 0.25% trypsin and the cell density was adjusted to 1x106 cells/ml. The cells were fixed and permeabilized with FIX & PERM Cell Permeabilization Kit (cat. no. GAS003; Thermo Fisher Scientific, Inc.) at 37˚C for 30 min. The cells were then incubated at 37˚C for 30 min with PBS and anti-vimentin antibody (1:100, cat. no. ab92547; Abcam), before being incubated with FITC-labeled secondary antibody (1:1000, cat. no. ab6717) at 37˚C for 30 min. Cells were then analyzed using a BD FACSCanto™ II flow cytometer (BD Biosciences) with FlowJo software (Version 7.6, BD Biosciences).
9
Immunofluorescence Staining of MC3T3-E1 Cells
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MC3T3-E1 cells cultured for 7 days were fixed with 4% paraformaldehyde in PBS for 20 min. The samples were permeabilized with 0.1% Triton-X 100 in phosphate buffer for 5 min. After being blocked using 1% BSA in phosphate buffer for 30 min, the samples were incubated with primary antibody (1:500, Abcam) for 60 min at room temperature. Subsequently, the samples were washed by PBS three times, followed by incubation in fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:500, Abcam) at ambient temperature in the dark for 60 min. Finally, the cell nuclei were dyed with 4′,6-diamidino-2-phenylindole (DAPI) for 1 min. Photos were taken on a confocal laser scanning microscope (LSM 780, ZEISS).
10
VE-Cadherin Immunofluorescence Staining
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The cells were rinsed with PBS and fixed with 4% paraformaldehyde. After being washed and sealed, the cells were treated with the anti-VE-cadherin antibody (Abcam, USA) overnight at 4°C. Then, the FITC-labeled secondary antibody (Abcam, USA) was applied. DAPI was used for nuclear staining, and the cells were photographed using the CKX53 microscope (Olympus, Japan).
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