For staining the SLC spheres and differentiated SLCs, the cells were fixed with 4% paraformaldehyde, permeabilized by 0.3% Triton X-100 (Sigma), and incubated with the indicated first antibodies and fluorescence-conjugated secondary antibodies (Molecular Probes, Life Technologies, CA) and stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) for nuclear staining. The stained cells were observed with a fluorescence microscope (Olympus, Japan). The antibodies used included anti-CYP24A1 (Abcam, UK) and anti-CAIX (Abcam, UK). fluorescence-conjugated secondary antibodies were from Life Technologies (CA, USA).
Fluorescence conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescence-conjugated secondary antibodies are laboratory reagents used in various immunoassay techniques, such as immunofluorescence and flow cytometry. These antibodies are designed to detect and visualize the binding of primary antibodies to their target antigens, providing a fluorescent signal that can be detected and analyzed using specialized laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (13 mentions)
Triton x 100, by Merck Group (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (10 mentions)
Fluorescence microscope, by Olympus (8 mentions)
Vectashield, by Vector Laboratories (4 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
Lsm 700, by Zeiss (3 mentions)
Confocal microscope, by Zeiss (3 mentions)
Triton x 100, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Ix70 microscope, by Olympus (3 mentions)
Lsm 510, by Zeiss (2 mentions)
Tcs sp8 sted, by Leica (2 mentions)
Fluoro gel with tes buffer, by Electron Microscopy Sciences (2 mentions)
Dapi nuclear stain, by Roche (2 mentions)
Prolongold antifade, by Thermo Fisher Scientific (2 mentions)
Goat serum, by Merck Group (2 mentions)
130 protocols using fluorescence conjugated secondary antibody
1
Immunofluorescence Staining of SLC Cells
2
Visualizing NLRP3 and ASC Specks in CTL-Stimulated DCs
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Cells were cultured on glass in complete medium. For staining ASC speck, LPS-primed and ova-pulsed BMDCs on coverslips were washed twice with PBS, incubated with CTLs that were pre-labelled with Alexa Fluor 647-CD8α (eBioscience) and fixed for 15 min at room temperature with 4% formaldehyde in PBS, and then were washed three times with PBS. After permeabilization with 0.1% Triton X-100 in PBS and blocking with 5% BSA in PBS for 1 h, cells were incubated with anti-NLRP3 (Cryo-2; AdipoGen) and anti-ASC (sc-22514-R; Santa Cruz Biotechnology, Inc.) primary antibodies (in 5% BSA) for 1 h at room temperature. Cells were washed three times with PBS and then were incubated for 1 h at room temperature with the appropriate fluorescence-conjugated secondary antibody (Invitrogen). The samples were washed three times in PBS, and then incubated with DAPI for 5 min. Samples
were observed with a Zeiss LSM510.
were observed with a Zeiss LSM510.
3
Immunofluorescence Analysis of Cell Markers
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Immunofluorescence analysis was performed as described previously [21] (link). Briefly, cells were fixed and incubated with the primary antibodies E-cadherin (610,181, 1:500, BD Biosciences) or Vimentin (610,193, 1:500, BD Biosciences) overnight at 4°C. After washing with PBS, cells were then incubated with fluorescence-conjugated secondary antibody (Invitrogen). Images were captured after staining with DAPI solution.
4
SSTR2 Immunocytochemistry Protocol
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Immunocytochemistry was performed as previously described [10 (link)]. Cells fixed in 4% paraformaldehyde were blocked with 3% bovine serum albumin for 30 min at room temperature (25–27°C) and incubated with SSTR2 primary antibody (clone UMBI; 1:500; Abcam, Cambridge, UK) at 4°C overnight. The cells were washed, incubated with the appropriate fluorescence-conjugated secondary antibody (1:200; Invitrogen) for 1 h at room temperature, and counterstained with Hoechst (Invitrogen). The coverslips were then mounted, and the cells were observed under a light microscope. Images were recorded using an Olympus BX53 microscope with Olympus Cell Sens software (Tokyo, Japan).
5
Immunofluorescent Localization of PARK2 and YAP
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Cells on the coverslips were fixed with 4% paraformaldehyde and incubated with the primary antibody against PARK2 (Santa cruz, sc-32282), YAP (CST, 14074) at 4 °C overnight. After washing with PBS, cells were then incubated with fluorescence-conjugated secondary antibody (Invitrogen, Carlsbad, CA), and subsequently counterstained with DAPI (Life Technology). Images were captured after staining with anti-fade DAPI solution using a confocal laser-scanning microscope (Leica TCS SP8 STED). The fluorescence-integrated density was measured by ImageJ software and the mander's co-localization coefficients were generated by Zen software.
6
Immunofluorescence Staining and ROS Analysis
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For immunofluorescence staining, the cells were fixed in 4% paraformaldehyde in phosphate-buffered solution (PBS) for 15 min, followed by three PBS washes. The fixed cells were incubated with 0.1% Triton X-100 in PBS for 5 min, blocked in 5% BSA, and incubated with HA probe antibody (sc-805, Santa Cruz Biotechnology) overnight before incubation with the fluorescence-conjugated secondary antibody (Invitrogen, USA) for 1 hour. Images were obtained using an Eclipse TE2000-U confocal laser scanning microscope (Nikon, Tokyo, Japan) after counterstaining with 4′,6-diamidino-2-phenylindole. The ROS levels were measured using a Reactive Oxygen Species Assay Kit (Beyotime, China). Flow cytometry analysis was performed according to the manufacturer’s protocols, and the results were obtained. Cell survival of the HTK cells post transfection with and without antioxidant (NAC, 0.1 mM, Sigma-Aldrich) was measured using WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, China).
7
Immunofluorescence Microscopy of Cells
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Cells were plated in Fisher 6-well chamber slides at a density of 20,000 cells/well. Twenty-four hours later, doxycycline was added and the cells were incubated for an additional 36 h. Indirect immunofluorescence was modified from [56 (link)]. Cells were incubated with the appropriate fluorescence-conjugated secondary antibody (Invitrogen). 4′,6-diamidino-2-phenylindole (DAPI; Boehringer-Mannheim) was used to stain the nuclei. Microscopy was performed on a Zeiss Axiovert 200 M, using Axiovision 4.4 and the ApoTome imaging system.
8
Lentiviral Infection Efficacy in AVG Neurons
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Efficacy of the lentivirus infection was assessed by immunofluorescence staining. Briefly, isolated AVGs from 3 rats with transfection of lentiviral rat Cav2.2‐α shRNA with pGFP were postfixed in 4% paraformaldehyde, followed by soaking of AVGs in 30% sucrose for 12 hours at 4°C for cryostat protection. AVGs were cut into 10‐μm‐thick sections at −20°C and then mounted on precoated glass slides. AVG sections were incubated with 10% donkey serum for 1 hour, followed by incubation with goat anti–choline acetyltransferase antibody (a cholinergic neuronal marker; EMD Millipore, Billerica, MA) overnight at 4°C. Then AVG sections were incubated with fluorescence‐conjugated secondary antibody (Invitrogen, Carlsbad, CA) for 1 hour at room temperature. AVG sections were examined under a Leica (Wetzlar, Germany) fluorescent microscope with corresponding filters for choline acetyltransferase (red color) and pGFP (green color). The imaging was captured by a digital camera system. Figure 1 B shows that microinjection of lentiviral rat Cav2.2‐α shRNA with pGFP into the AVG induced pGFP expression in almost all choline acetyltransferase–positive AVG neurons (n=3 rats), confirming the efficacy of virus infection and proper microinjection into the AVG.
9
Nuclear Translocation of p65 and NFATc1 in Osteoclastogenesis
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To determine p65 nuclear translocation, BMMs were treated with 100 μmol/L STA for 4 hours, followed by stimulation with 50 ng/mL RANKL for 15 minutes. For determining NFATc1 nuclear translocation, BMMs were stimulated with 25 ng/mL M‐CSF and 50 ng/mL RANKL in the presence or absence of 100 μmol/L STA for 0, 2 or 4 days. The cells were fixed with 4% PFA for 15 minutes, permeabilized with 0.3% Triton X‐100 for 20 minutes and non‐specifically blocked in 1% BSA for 30 minutes. The fixed cells were washed and incubated overnight with the primary antibody against NFATc1 or p65. Next, the cells were incubated with an appropriate fluorescence‐conjugated secondary antibody (Invitrogen) for 120 minutes, stained with rhodamine‐conjugated phalloidin for 20 minutes and mounted in Fluoroshield with DAPI (Sigma‐Aldrich). Fluorescent images were captured using a fluorescence microscope (Leica).
10
Quantifying Neuronal Morphology Using Immunofluorescence
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