Animals were bled from the tail one day before T. vaginalis challenge. Sera was collected and stored at -80 °C until use. Parasites at logarithmic-phase growth were collected by centrifugation and washed with PBS three times. Half a million cells in 100 μl were added to poly-L-lysine pre-coated slides and air-dried at room temperature for 20 min. After fixation with pre-cooled methanol, the slides were rehydrated with PBS for 20 min and then incubated with anti-sera (1:2,000) collected from mice with or without immunization for one hour. After extensive washes with PBS, another 45 min incubation was undertaken with goat-anti mouse IgG FITC (1:800; Invitrogen) and anti-fade mounting medium with DAPI (50 μg/ml) was conducted. Photos were taken under a fluorescent microscope (Zeiss, Oberkochen, Germany).
Goat anti mouse igg fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-mouse IgG-FITC is a secondary antibody conjugated with fluorescein isothiocyanate (FITC). It is designed to detect and visualize mouse immunoglobulin G (IgG) in immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva, by BD (2 mentions)
Facscan system, by BD (2 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Fitc goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Fluorescent microscope, by Zeiss (1 mentions)
Facscelesta, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg pe, by Thermo Fisher Scientific (1 mentions)
Ssea4, by Cell Signaling Technology (1 mentions)
Anti cd14 fitc, by BD (1 mentions)
Anti cd206 pe, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Kaluza, by Beckman Coulter (1 mentions)
Tcs sp5, by Leica (1 mentions)
Ddh2o, by Merck Group (1 mentions)
Ab18311, by Abcam (1 mentions)
H 1200, by Vector Laboratories (1 mentions)
Facscan, by BD (1 mentions)
Dhr123, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
14 protocols using goat anti mouse igg fitc
1
Immunofluorescence Assay for T. vaginalis
2
Immunofluorescent Labeling of Collagen I
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Mouse Collagen I Monoclonal Antibody (catalog CSI 008-01-02) and goat anti-mouse IgG-FITC (catalog sc-2010) antibodies were purchased from Invitrogen and Santa Cruz Biotechnology, respectively, and were used without further purification. Lyophilized PPMS and Col-PPMS were first dissolved in PBS and blocked with 5% bovine serum albumin (BSA) for 1 h at 4°C. Then the particles were washed with Tris buffer saline with 0.1% v/v tween-20 (TBST) before incubation with Collagen I monoclonal antibody (dilution 1:1000) overnight at 4°C. Next, the particles were washed with TBST 3–4 times and incubated with secondary antibody (dilution 1:2500) for 4 h followed by washing with TBST. At each step, particles were collected via centrifugation at 100 g in for 5 min. Finally, Col-PPMS and PPMS were observed under Nikon CLSM with bright field and FITC channels.
3
Quantifying Protein Expression by Flow Cytometry
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For flow cytometry studies of protein marker expression, cells were dissociated into single cells by incubating them with 0.25% trypsin (Gibco) for 5 min at 37 °C and washed with 1x PBS twice. The dissociated cells were fixed with 75% ethanol at 4 °C overnight. Then, the cells were permeabilized with 0.05% Triton X-100 for 3 min and rinsed with 1x PBS twice. The cell number was adjusted to 1 × 106 cells per tube in 700 μL of 1x PBS for each protein marker. The cells were incubated with primary antibodies including: OCT-4 (1:500 dilution, Cell Signaling Technologies), SSEA-4 (1:500 dilution, Cell Signaling Technologies), TUJ-1 (1:500; R&D Systems, Minneapolis, MN, USA), or cTnT (1:500 dilution, Cell Signaling Technologies) at 4 °C overnight. Afterwards, the samples were rinsed with 1x PBS thrice before incubation with secondary antibodies (goat anti-mouse IgG FITC and goat anti-rabbit IgG PE, Invitrogen) at 1:1000 dilution for 1 h at RT. The samples were then washed with 1x PBS twice before analysis using a BD FACSCelesta (Franklin Lakes, NJ, USA) flow cytometer. The cells incubated with secondary antibody but no primary antibodies were processed and washed in the same way for analysis to serve as the negative/isotype control. The resultant data was analyzed with the BD Flowjo software (v10).
4
Quantifying DENV Infection in Macrophages
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Expression of macrophage surface and intracellular molecules was evaluated by flow cytometry. Intracellular staining using anti-CD68-PE (BD Pharmingen) and surface staining using anti-CD14-FITC (BD Pharmingen), CD83-PeCy-7 (eBioscience) and anti-CD206-PE (BD Pharmingen) were performed on MDMs and D3-MDMs. For each experiment, unstained and isotype controls were included. DENV infection was measured by intracellular detection of the E protein using the murine monoclonal 4G2 antibody (Millipore, Darmstadt, Germany) and a secondary antibody, the goat anti-mouse IgG-FITC (Invitrogen). Unstained cells, mock-infected cells plus secondary antibody only, mock-infected plus detection pair and infected cells plus secondary antibody were included as controls for every experiment. The percentage of infected cells was expressed as number of 4G2 positive cells over the total number of cells analyzed. Surface and intracellular staining samples were read on a FACScanto flow cytometer (BD Biosciences) and data were analyzed using the FACSdiva (BD Biosciences) and Kaluza (Beckman Coulter) softwares.
5
Flow Cytometric Immunophenotyping of Lymph Node Cells
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Cell treatment was performed according to an established procedure (Razzuoli et al., 2012 (link)), with minor modifications. Briefly, frozen lymph nodes cells were thawed at 38°C and washed with FACS-Buffer (0.1% sodium azide + 2% fetal calf serum in PBS). Then, they were divided into aliquots (106 cells each) and reacted with monoclonal antibody (mAb) CD21 (Southern Biotech, cat. 4530-02), Mil-2 (AbCAM, cat. 23919-1), PMN (AbD Serotec, cat. MCA2599F), or FACS buffer only (control) for 30 min at 4°C, respectively. Cells were washed, and again incubated for 30 min at 4°C in FACS buffer containing goat anti-mouse IgG-FITC (Invitrogen, Molecular Probes®, cat: A10683). After washing in FACS buffer, cells were resuspended in 100 μL of the same buffer and 1:4 diluted. Samples were analyzed in a GUAVA MILLIPORE flow cytometer (Millipore Software). The typical forward and side scatter gate was set to exclude dead cells from the analysis. The percentage of positive cells beyond the threshold fluorescence channel was assessed in each sample on 10,000 events and compared between mAb-treated and control cells. For each antibody, results were expressed in terms of net percentage of positive cells.
6
Quantifying DNA Damage Response in U2OS Cells
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U2OS cells were grown on coverslips at low density (5 × 104 cells/mL) in 6-well plates and transfected with the siControl, siDNAH2, or siFANCA, respectively. After the treatment of MMC (80 ng/mL in ddH2O, Sigma) or ddH2O for 24 h, cells were fixed with 4% PFA in PBS for 10 min and permeabilized with 0.1% Triton X-100 and 0.5% NP-40 in PBS for 15 min at room temperature. Cells were incubated with primary antibody overnight at 4°C, the secondary antibody for 1 hour, and DAPI (Vector H-1200) dihydrochloride for 10 min at room temperature. The images were obtained using a confocal microscope (Leica TCS SP5). The primary antibody: mouse anti-FANCD2 (FI17, sc-20022, Santa Cruz), mouse anti-γH2AX (ab18311, Abcam). The secondary antibody: goat anti-mouse IgG-FITC (Invitrogen 62-6312), goat anti-mouse IgG-CY3 (EarthOx E031610).
7
CD22 Expression and Antibody Binding
8
Flow Cytometry for DENV Infection
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Flow cytometry was used to assess the frequency of DENV-infected cells [16 (link),17 ]. Briefly, DENV infection was evaluated through the intracellular detection of DENV E antigen at 24 hpi fixing the cells with fixation/permeabilization buffer (eBioscience, USA). Following washing steps with PBS, cells were stained with the monoclonal antibody 4G2 (Millipore, Germany) for 40 min, followed by 40 min staining with goat anti-mouse IgG-FITC (Thermo Scientific, USA). All data acquisition and analysis were done using the BD FACScan system and FACSDiva software, respectively.
9
Analyzing DENV Infection and Immunomodulation
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Flow cytometry was used to assess the frequency of DENV-infected cells, expression levels of VDR and, changes of TLR3/4/9 expression, as previously described [17 (link),18 (link)]. Flow cytometry was also used for assessing the production of ROS production by the detection of the dihydrorhodamine 123 (DHR 123; Sigma Aldrich, USA). DENV infection was evaluated through the intracellular detection of DENV E antigen at the indicated time points and the cells were fixed using a fixation/permeabilization buffer (eBioscience, USA). Following washing steps with PBS, cells were stained with the monoclonal antibody, 4G2 (Millipore, Germany) for 40 min, followed by 40 min staining with goat anti-mouse IgG-FITC (Thermo Scientific, USA). Expression of TLR3, TLR4, and TLR9 in DENV-2-infected and mock-infected cells was evaluated at 2, 8, and 24 hpi as we described in [25 (link)]. All data acquisition and analysis were done using the BD FACScan system and FACSDiva software, respectively.
10
Osteocalcin Expression Analysis in Rabbit Tibiae
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Prior to the sacrifice of the rabbit tibiae, the implants were removed from each tibia and, along with the surrounding bone, the specimens were preserved for 3 h in the refrigerator in the RPMI media, which contained penicillin (50 U/mL) and streptomycin (50 μg/mL). The cells underwent immunostaining and were incubated for 15 min with a protein block (DAKO, Agilent, Santa Clara, CA, USA, X0909) to remove non-specific binding protein. The cells were then incubated for 30 min with a diluted osteocalcin primary antibody (1:100 dilution in 3% bovine serum albumin (BSA), #MA120788, Thermo Fisher Scientific, USA). After being rinsed in PBS, these cells were incubated for 1 h with a diluted secondary antibody (1:200 diluted goat anti-mouse IgG-FITC in 3% BSA, #A10530, Thermo Fisher Scientific, Waltham, MA, USA) in a dark room and washed with PBS. Subsequently, nuclear counterstaining was performed using Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) (1:10,000 dilution) for 5 min. After the counterstaining, the images were obtained by fluorescence microscopy using Axio Observer.A1 (Carl Zeiss, Jena, Germany).
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