Cytoskeleton changes and BrdU incorporation were analyzed by IF as reported20 (link),28 (link). Endogenous AR was visualized using diluted (1:100) rabbit polyclonal antibody against the N-terminal domain of AR (Ab-2; Neomarkers; Portsmouth, New Hampshire, USA). Diluted (1:200 in PBS containing 0.2% bovine serum albumin) anti-rabbit fluorescein isothiocyanate-conjugated antibody (Jackson Laboratories, Bar Harbor, Maine, USA) was used as a secondary reagent. Nuclei were stained with Hoechst 33258 (Sigma-Aldrich) and coverslips were inverted and mounted in Mowiol (Sigma-Aldrich). In AR/FlnA co-localization and AR nuclear translocation analysis, the receptor was revealed using the rabbit Alexa Fluor 488-conjugate anti AR antibody (clone D6F11 from Cell Signaling; Danver, MA, USA). FlnA was stained using diluted (1:50 in PBS) goat polyclonal anti-FlnA antibody (Ab11074; Abcam; Cambridge, UK). Goat antibody was detected using diluted (1:300 in PBS) rabbit anti-goat Texas red-conjugated antibody (Abcam). Cells on coverslips were analyzed using a DMLB (Leica; Wetzlar, Germany) fluorescent microscope, equipped with HCX PL Apo ×63 oil and HCX PL Fluotar ×100 oil objectives. Images were captured using DC480 camera (Leica) and acquired using Leica Suite software. AR/FlnA co-localization was analyzed29 (link), using a Zeiss (Oberkochen, Germany) LSM 510 laser scanning confocal microscope.
Lsm 510 confocal laser scanning microscope
Manufactured by Zeiss
Sourced in Germany, United States, Japan
The LSM 510 is a confocal laser scanning microscope manufactured by Zeiss. It is designed to capture high-resolution images of samples by scanning them with a focused laser beam and detecting the emitted fluorescence or reflected light. The system utilizes a set of optical components, including lenses, mirrors, and detectors, to create a detailed, three-dimensional representation of the sample.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (8 mentions)
Vectashield, by Vector Laboratories (7 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (5 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Uncoated glass bottom petri dishes, by MatTek (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Oct4 c 10, by Santa Cruz Biotechnology (3 mentions)
Lsm 510, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole dapi mounting medium, by Merck Group (3 mentions)
Lsm image browser, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Isolution dt 36, by Zeiss (3 mentions)
Fluorescent mounting medium, by Agilent Technologies (3 mentions)
Glass bottom dishes, by MatTek (2 mentions)
Axiophot fluorescence microscope, by Zeiss (2 mentions)
302 protocols using lsm 510 confocal laser scanning microscope
1
Immunofluorescence Analysis of Cytoskeleton and AR
2
Visualizing Actin Cytoskeleton Reorganization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Actin cytoskeleton reorganization was assessed via filamentous actin (F-actin) staining, as reported previously [19 (link)]. Briefly, after each treatment, the cells were fixed with 4% paraformaldehyde for 24 hours and permeabilized with 0.2% Triton X-100. Following blocking of nonspecific binding using 0.5% foetal bovine serum, the cells were stained with rhodamine-conjugated phalloidin (100 ng/ml, Biotium, USA) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 5 μg/mL, Sigma) for 20 minutes. Images of the fluorescently labelled specimens were captured using an LSM 510 laser scanning confocal microscope equipped with immersion lenses (Carl Zeiss Meditec, Jena, Germany). The stress fibres in 100 cells on each slide were randomly analysed under a Zeiss fluorescence microscope. Cells exhibiting stress fibres were analysed with an Olympus fluorescence microscope. LSM 510 software was employed to determine the numbers of cells exhibiting stress fibres according to previous studies [20 (link),21 (link)]. The data were expressed as the percentages (mean ± SD) of 100 cells counted on each slide.
3
Evaluating Phagocytosis and Trafficking of Bacteria
4
Live-cell Imaging of Focal Adhesions
5
Live Imaging of Focal Adhesion Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GFP-paxillin-expressing cells were plated on glass-bottom dishes (MatTek) coated with 1 μg ml−1 FN and allowed to adhere for 1 h. Images were acquired with an LSM 510 laser scanning confocal microscope (CarlZeiss) equipped with a × 63/1.40 NA Plan Apo oil objective lens at 37 °C with 5% CO2. Images were taken every 30 s for 45 min. The rate constants for assembly and disassembly of adhesions were measured through the Focal Adhesion Analysis Server49 (link).
6
Immunofluorescence Analysis of Mitotic Spindle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells and H460 cells were transfected with PIG3 or NC siRNAs. Cells were plated and cultured on poly-d-lysine-coated cover slides 48 h after transfection. Cells were washed twice in PBS and fixed in 4% paraformaldehyde/PBS at room temperature for 30 min. Next, the cells were permeabilized with 0.5% Triton X-100/PBS at room temperature for 15 min. After permeabilization, cells were blocked with 1% bovine serum albumin/PBS at room temperature for 30 min. Immunostaining was performed by incubating with anti-α-tubulin antibody, γ-tubulin (Sigma, St Louis, MO, USA) and phosphorylated H3 (pSer10) (Cell Signaling Technology, Beverly, MA, USA) antibodies (1:1000) for 4 h at room temperature. After incubating with the primary antibodies, cells were washed three times with PBS. Cells were then incubated with Alexa-488 conjugated anti-rabbit and Alexa-568 conjugated anti-mouse secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. For visualization of DNA, 4, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) was added to the mounting medium. Images were obtained using a LSM 510 laser-scanning confocal microscope (Zeiss, Germany).
7
Visualizing Burkholderia pseudomallei Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
J774A.1 or HeLa cells were infected with B. pseudomallei wild-type or its derivatives at a multiplicity of infection (MOI) of 0.5 or 50, respectively. At the experimental end-point, the infected cells were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) overnight. Cells were permeabilized with PBS containing 0.1% (v/v) Triton X-100 for 30 min and incubated with PBS containing 1% (w/v) bovine serum albumin (BSA) for 30 min at room temperature. Bacteria were stained with rabbit polyclonal anti-B. pseudomallei lipopolysaccharide antibody (kindly provided by Prof. R.W. Titball, Exeter University, UK) at 37°C for 1 h, washed with PBS and then incubated with goat anti-rabbit antibody-Alexa Fluor488 (Molecular Probes). Nuclei were stained with DAPI (4', 6-Diamidino-2-Phenylindole, Dihydrochloride; Molecular Probes). F-actin was directly stained using phalloidin568 (Molecular Probes). BimA protein was detected with a panel of three previously described monoclonal antibodies (Stevens et al., 2005b (link)). Bound anti-BimA was detected with goat anti-mouse antibody-AlexaFluor568 (Molecular Probes). Images were captured using an LSM 510 laser scanning confocal microscope (Carl Zeiss).
8
Subcellular Localization of GmPGL2 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-length CDS and the 594-bp region (GmPGL21-198) of the GmPGL2 gene were fused to green fluorescent protein (GFP) at the C terminal, and then amplified and cloned into the modified 3301H vector at the XmaI and HindIII sites. We prepared two constructs, namely, 35:GmPGL2-GFP and 35S:GmPGL21-198-GFP, which were introduced into A. tumefaciens (strain EHA105) and subsequently used to infiltrate Nicotiana benthamiana leaves as described previously (Waadt and Kudla, 2008 (link)). The GFP fluorescence signals were detected using a LSM510 laser scanning confocal microscope (Carl Zeiss, Germany). Transmission electron microscopy was performed according to a previously described method (Kwon and Cho, 2008 (link)).
9
Multimodal Imaging for 3D Modeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Any microscopy imaging modality that creates 3D datasets can be used for the creation of physical models. This includes microscopy techniques like confocal microscopy [11 (link)] and multiphoton microscopy [12 (link)] that section tissue by taking 2D images at different depths through the sample, as well as optical techniques that are tomographic, like optical projection tomography (OPT) [13 (link)] and electron tomography [14 (link)] that use 2D projections at many different angles to create a 3D dataset. The imaging modalities used to collect the three independent datasets for the models described here are confocal microscopy [11 (link)], electron tomography [14 (link)] and multiphoton microscopy [15 (link)].
Specifically, a Caenorhabditis elegans embryo expressing a membrane-localized green fluorescent protein (GFP) marker was imaged using multiphoton laser scanning microscopy [16 (link)]. A C. elegans distal tip cell expressing a GFP marker was imaged with 63X 1.4NA objective and 488 nm excitation on a Zeiss LSM510 laser scanning confocal microscope. And a prevacuolar compartment of a high-pressure frozen/freeze substituted maize aleurone cell was imaged by dual electron tomography in a Tecnai F30 transmission electron microscope [17 (link)].
Specifically, a Caenorhabditis elegans embryo expressing a membrane-localized green fluorescent protein (GFP) marker was imaged using multiphoton laser scanning microscopy [16 (link)]. A C. elegans distal tip cell expressing a GFP marker was imaged with 63X 1.4NA objective and 488 nm excitation on a Zeiss LSM510 laser scanning confocal microscope. And a prevacuolar compartment of a high-pressure frozen/freeze substituted maize aleurone cell was imaged by dual electron tomography in a Tecnai F30 transmission electron microscope [17 (link)].
10
Immunocytochemistry Analysis of Cellular Proteins
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!