Cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilised with 0.01% Triton X-100 for 15 minutes. The cells were then incubated with the desired primary antibodies at 37°C for 1 hour, followed by species-specific secondary antibodies at 37°C for 1 hour. In the growth kinetics studies, the samples were probed with primary antibodies against Alphaviruses (Santa Cruz sc-58088, 1∶250 dilution), followed by FITC-conjugated secondary antibodies (Millipore, 1∶500 dilution). In the polarized infection studies, the samples were co-labeled with primary antibodies against CHIKV E2 glycoproteins (1∶100 dilution) and FITC-conjugated secondary antibodies (Millipore, 1∶250 dilution) together with primary antibodies against ZO-1 proteins (Invitrogen, 1∶500 dilution) and Dylight-594-conjugated secondary antibodies (Thermo Scientific, 1∶100 dilution). In the virus binding assay, the samples were probed with primary antibodies against Alphaviruses (Santa Cruz, 1∶250 dilution), followed by FITC-conjugated secondary antibodies (Millipore, 1∶500 dilution). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 20 minutes. 1×PBS washes were performed after each incubation step. The samples were subsequently mounted onto glass slides using DABCO and viewed under the Olympus IX81 inverted microscope or Olympus FV1000 confocal microscope.
Fitc conjugated secondary antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom
FITC-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of a fluorescent dye, fluorescein isothiocyanate (FITC), which is covalently attached to a secondary antibody. The secondary antibody is designed to bind to the primary antibody, which in turn binds to the target antigen, allowing for the detection and visualization of the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Nunc lab tek chamber, by Thermo Fisher Scientific (3 mentions)
Eclipse ti, by Nikon (3 mentions)
Cy3 conjugated secondary antibody, by Merck Group (2 mentions)
Anti β catenin antibody, by BD (2 mentions)
Axio observer, by Zeiss (2 mentions)
Zen 3, by Zeiss (2 mentions)
Facscan flow cytometer, by BD (2 mentions)
Mouseanti γh2ax specific antibody clone jbw301, by Merck Group (2 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Primary antibody against cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Axio observer microscope, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
H3k9me2, by Sino Biological (1 mentions)
H3k27me3, by Sino Biological (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Digital camera, by Nikon (1 mentions)
Nis elements ar 3, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
62 protocols using fitc conjugated secondary antibody
1
Immunofluorescence Staining of Alphavirus Infection
2
Immunohistochemical Analysis of Muscle Cell Infiltration and Capillary Density
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The 5-μm-thick paraffin sections were cut and fluorescent immunohistochemical analysis of sections was performed as previously described [24 (link)]. Cell infiltration in the skeletal muscles was examined by using anti-mouse Mac-3 antibody (1:200; Cedarlane Labs, Burlington, ON, Canada) overnight at 4 °C followed by incubation with a Cy3-conjugated secondary antibody (1:500; Chemicon International, Temecula, CA, USA). The slides were then washed in PBS and stained with anti-sarcomeric α-actin antibody (1:200; Abcam Inc. Cambridge, MA, USA) and incubated with an FITC-conjugated secondary antibody (1:500; Chemicon International). The nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA, USA). Capillary density from wild type and ECKO mice was assessed by immunostaining with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) antibody (1:200, LifeSpan BioSciences, Nottingham, UK) overnight at 4 °C and then with a FITC-conjugated secondary antibody (Chemicon International). Digital images were captured with a Nikon digital camera mounted directly onto a fluorescence microscopy. Images were analyzed with computerized imaging software (NIS-Elements AR 3.10, Nikon Instruments Inc., Melville, NY, USA) as previously described [24 (link)].
3
Immunofluorescence Staining Protocol for Stem Cell Markers
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The immunofluorescence staining was conducted as previously reported [55 (link)]. Detailed information for the antibodies are : TET1 (1:300, GeneTex, California, USA); 5hmC (1:500; Active Motif, California, USA); OCT-4 (1:300; Chemicon, Massachusetts, USA); stage-specific embryonic antigen 1 (SSEA-1; 1:200; Chemicon, Massachusetts, USA); H3K9me2 (1:500; Sino Biological Inc., Beijing, China); H3K27me3 (1:500; Sino Biological Inc., Beijing, China); Sox2 (1:200; Chemicon, Massachusetts, USA); proliferating cell nuclear antigen (PCNA; 1:200; Millipore, Massachusetts, USA); 5-bromo-2′-deoxyuridine (BrdU; 1:300; Santa Cruz, California, USA), FITC-conjugated secondary antibody (1:500; Chemicon, Massachusetts, USA), HOECHST33342 (Sigma-Aldrich, Missouri, USA). The immunofluoresence intensity was analyzed by ImageJ software (National Institutes of Health, USA).
4
Immunofluorescence Staining of HER2 in RT4 Cells
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RT4 cells were cyto‐spinned on glass slides, and washed once with PBS, fixed in PBS containing 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilized with 0.2% Triton X‐100 for 10 min and incubated with PBS containing 4% BSA (Sigma; St. Louis, MO, USA) for 30 min. Incubations were performed with HER2 primary antibody diluted in PBS containing 1% BSA for 1 h at room temperature. Afterwards, cover slides were washed and incubated for 30 min with the fluorescein isothiocyanate (FITC)‐conjugated secondary antibody (CHEMICON, Billerica, MA, USA). Fluorescence images were collected on a Zeiss Axioplan.
5
Mesenchymal Origin of Bone Marrow Stromal Cells
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To investigate the mesenchymal origin of BMSCs at second passage, 5000 cells were cast equally into each 6-well culture plate. The immunocytochemistry stages were performed as previously mentioned (16) . In summary, the cells were placed in paraformaldehyde solution 4 % for 20 minutes and after being washed with phosphate buffered, the cells were placed in 0.3 % Triton X for 15 minutes. After being washed with PBS, the cells were exposed to the primary antibody for 24 hours at 4 °C. The primary antibodies included CD31 (endothelial cells marker), fi bronectin (mesenchymal stem cells marker), CD34 (hematopoietic stem cells marker) and P62 (autophagy marker) from ABCAM Company. After the primary antibody incubation, the cells were washed with PBS and then FITC-conjugated secondary antibody (1:100; Chemicon) was added for 2 hours at room temperature. The cells were counted using propidium iodide (PI) through which the nucleus of the cells became red. For assessing autophagy, the BMSCs were immunelabelled with a primary p62 antibody, incubated with the FITCconjugated secondary antibody (shown in green), and a primary LC3II antibody, incubated with Alexa fl our secondary antibody (shown in red). The number of autophagosome immune-reactive cells was determined to estimate the autophagy activity.
6
Microtubule Network Visualization in U-87 MG Cells
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U-87 MG cells were seeded (10 000
cells per well, 300 μL)
on eight-well chamber slides (Lab-Tek). After 24 h, the medium was
removed, and the cells were treated with FONPs–PTX (181 or
362 μg/mL of FONPs–PTX corresponding to 5 or 10 μM
PTX-equivalent concentration in FONPs–PTX) for 24 h. After
drug treatment, immunofluorescence staining of the microtubular network
was performed using anti-β-tubulin primary antibody (1:200,
mouse monoclonal, Sigma-Aldrich) and FITC-conjugated secondary antibody
(1:200, Sigma-Aldrich) as previously described.9 (link) To double-label nuclei, cells were further stained with
4,6-diamino-2-phenylindole (DAPI) (0.25 μg/mL). Cells were observed
with an epifluorescence microscope (Leica DM-IRBE), 40× objective
lens, coupled to a digital camera (Coolsnap FX, Princeton Instruments).
cells per well, 300 μL)
on eight-well chamber slides (Lab-Tek). After 24 h, the medium was
removed, and the cells were treated with FONPs–PTX (181 or
362 μg/mL of FONPs–PTX corresponding to 5 or 10 μM
PTX-equivalent concentration in FONPs–PTX) for 24 h. After
drug treatment, immunofluorescence staining of the microtubular network
was performed using anti-β-tubulin primary antibody (1:200,
mouse monoclonal, Sigma-Aldrich) and FITC-conjugated secondary antibody
(1:200, Sigma-Aldrich) as previously described.9 (link) To double-label nuclei, cells were further stained with
4,6-diamino-2-phenylindole (DAPI) (0.25 μg/mL). Cells were observed
with an epifluorescence microscope (Leica DM-IRBE), 40× objective
lens, coupled to a digital camera (Coolsnap FX, Princeton Instruments).
7
Immunofluorescence Staining of β-catenin
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The sample preparation has been described previously [31 (link)]. Briefly, drug-treated HCT 116 cells on coverslips were fixed in freshly-prepared 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA (Bovine Serum Albumin, BSA) in PBS (phosphate buffer saline, PBS). Afterwards, the cells were incubated with an anti-β-catenin antibody (BD Biosciences) overnight at 4 °C. Following 3 times washing with PBS, a FITC conjugated secondary antibody (Sigma-Aldrich) was employed for 1 h incubation at room temperature. Ten minutes prior to the next washing step, 4’,6-diamidino-2-phenylindole (DAPI) was added for nucleus staining. After washing and mounting procedures, the fluorescent images were examined under a microscope (Nikon, Tokyo, Japan).
8
Quantification of Viral Infection by Flow Cytometry
9
Cell Apoptosis Pathway Evaluation
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BP (Sigma-Aldrich) was dissolved in vitamin K solution (Sigma-Aldrich). The stock solution was with a concentration of 200 μg/μl. The following antibodies were used: caspase 3, 7, 8, 9 (Cell Signaling Technology, Danvers, MA, USA); tubulin and beta-actin (Abcam, Cambridge, UK). The secondary antibodies goat anti-rabbit and -mouse were purchased from Cell Signaling Technology. The FITC conjugated secondary antibody was purchased from Sigma.
10
Detect Exogenous HA-Tagged PGRMC1
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To detect the expression of exogenous HA tagged PGRMC1 in Fig. 1 e, cells were seeded on coverslips on a six well plate. The cells were washed with ice-cold PBS, mildly fixed with 3.7% formaldehyde for 5 min at 4 °C. The cells were then permeabilized with ice-cold 100% methanol for 10 min at − 20 °C, followed by overnight incubation with anti-HA tag antibody (Sigma, H3663). The cells were washed extensively and incubated with FITC conjugated secondary antibody (Sigma, F8521) in dark for 1 h at 4 °C. Cells were washed three times with PBS and counterstained with DAPI mounting solution. Images were captured using a Nikon Ti Eclipse Confocal microscope (Nikon Australia Pty Ltd).
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