Cells were seeded on coverslips coated with gelatin at an appropriate density. Cells were fixed by 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature and blocked with the blocking buffer (1X PBS, 0.2% Triton, 3% BSA) for 30 min. Cells were then incubated with the primary antibody for 2-3 h at room temperature, washed three times with 0.2% Triton X-100 in PBS, then incubated with fluorescence-labeled secondary antibodies (Invitrogen, 1:500 dilution) for 30 min at room temperature and rewashed three times. A drop (10 μL) of VECTASHIELD antifade mounting medium containing 4′,6′-diamidino-2-phenylindole (DAPI) (Vector labs, H-1200) was added on a labeled microscope slide. The coverslip was then placed on the drop, observed and photographed under the Carl Zeiss LSM 710 confocal fluorescence microscope.
Lsm710 confocal fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss LSM710 is a high-performance confocal fluorescence microscope. It is designed to provide high-resolution imaging of fluorescently labeled samples. The microscope uses a laser-scanning system to excite and detect fluorescence, enabling optical sectioning and 3D imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Dmi6000b microscope, by Leica (2 mentions)
Hoescht 33342, by Thermo Fisher Scientific (2 mentions)
Fluorescence labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Wga alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab73400, by Abcam (1 mentions)
Ab7760, by Abcam (1 mentions)
Ab10988, by Abcam (1 mentions)
Alexa 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Mildform, by Fujifilm (1 mentions)
Alexa 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Tissue studio, by Definiens (1 mentions)
Histofine mouse stain kit, by Nichirei Biosciences (1 mentions)
Paraffin, by Sakura Finetek (1 mentions)
24 protocols using lsm710 confocal fluorescence microscope
1
Immunofluorescence Staining of Cells
2
Inhibition of miR-96-5p Modulates ZEN-Induced Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TM3 cells were transfected with miR-96-5p inhibitor (10 nM) for 6 h followed by incubation with ZEN (50 µM) at 37˚C for an additional 48 h. Following treatment, the cells were fixed in 4% paraformaldehyde for 30 min at 4˚C, followed by permeabilization with 0.1% Triton X-100 (Sigma-Aldrich; Merck KGaA) at 4˚C and blocking with 5% bovine serum album (BSA; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Subsequently, the cells were incubated with the primary antibodies overnight at 4˚C. The primary antibodies used were as follows: Ki67 (1:200; cat. no. ab15580; Abcam) and LC3 (1:200; cat. no. ab192890; Abcam). The following morning, the cells were washed with PBS and incubated with goat anti-rabbit secondary antibodies (1:500; cat. no. ab7090; Abcam) for 1 h at room temperature. Subsequently, the cells were counterstained with 10 mg/ml DAPI fluorescence for 2 min to detect the nuclei. The stained cells were visualized and mounted using a LSM710 confocal fluorescence microscope (Carl Zeiss AG; magnification, x200).
3
Trehalose Rescue of Hypoxia-Induced Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections were obtained from the thoracic muscle of 5-day-old male locusts. The locusts were first subjected to severe hypoxia (2% Po2) for 6 h. In the trehalose rescue group, locusts were injected with 500 µg of trehalose solution (100 µg µl−1 in ddH2O) before hypoxic treatment. TUNEL staining was performed with the In Situ Cell Death Detection Kit (Roche, 11684817910, USA) following the manufacturer’s instructions. For details, thoracic muscle tissue sections were fixed with 4% paraformaldehyde for 20 min and washed with PBS for 30 min. The fixed tissue sections were then permeabilized with 0.1% TritonX-100 for 10 min at RT and treated with 3 U ml−1 DNase1 for 10 min to break the DNA strands. The tissue sections were then sequentially incubated with a TNNEL reaction mixture and 0.5% Hoechst (Life, H3570, USA) before washing with PBS three times. The tissue sections were imaged using an LSM 710 confocal fluorescence microscope (Zeiss) at a ×10 magnification. The experiments were repeated at least five times. TUNEL intensity was measured and quantified using Image J.
4
Immunodetection of βCBP in Integument
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
βCBP was detected in the integument via immunohistochemistry. The integument was fixed in 4% paraformaldehyde overnight. Paraffin-embedded integument tissue slides (5 µm thick) were deparaffinized in xylene and rehydrated with an ethanol gradient. The samples were blocked with 5% BSA for 30 min and then incubated with an anti-βCBP antiserum (1: 200) for 2 hr. The slides were washed and incubated for 1 hr with Alexa Fluor-488 goat anti-rabbit secondary antibody (Life Technologies). Hoechst (1: 500) was used for nuclei staining. The βCBP signals were detected using an LSM 710 confocal fluorescence microscope (Zeiss).
5
Cellular Response to DNPs Exposure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H1355 cell line (20 × 103 cells/coverslip) was plated on 10-mm glass coverslips placed on the bottom of 24-well plate, allowed to attach for 24 h under normal cell culture conditions, and then incubated with increasing DNPs* concentration (5, 10, 15 μg/mL) for 24 h. As negative control, the last supernatant obtained from nanoparticles labeling procedure was added to the cells. Cell nuclei and membranes were then stained with Hoechst 33342 (Invitrogen, Carlslab, CA, USA) and WGA-Alexa Fluor 488, respectively. Images were acquired at × 63 magnification on a LSM710 confocal fluorescence microscope (Carl Zeiss Inc., Peabody, MA, USA) with the appropriate filters. Cell fluorescence intensity was analyzed by using ImageJ software (http://imagej.nih.gov/ij/ ).
6
Quantifying Pancreatic Insulin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated mouse pancreata were fixed using Mildform (Wako) overnight at 4°C and embedded in paraffin (Sakura Finetek Japan). Sections of 4 μm were treated with 0.2% Triton-X100/PBS for 5 min and incubated with boiled 0.01 M citrate buffer, pH 6.0, for 20 min. Prior to application of primary antibody, sections were treated with Blocking Solution A from the Histofine MOUSESTAIN kit (Nichirei). Primary antibodies, including anti-OPG rabbit polyclonal antibody at 5.0 μg/ml (ab73400, abcam), anti-insulin mouse monoclonal antibody IN-05 at 1.0 μg/ml (ab7760, abcam), and anti-glucagon mouse monoclonal antibody K79bB10 at 4.4 μg/ml (ab10988, abcam) were applied overnight at 4°C. After washing with PBS, cells were stained with Alexa 488-conjugated goat anti-mouse IgG or Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes) for 1 hr at room temperature. After washing, sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and observed under an LSM710 confocal fluorescence microscope (Carl Zeiss) to determine signal localization or under a DMi6000B microscope (Leica) to measure signal intensity. The average signal intensity was measured by freehand region of interest (ROI) using Tissue Studio (Definiens) software to quantify insulin expression levels in pancreatic β-cells.
7
Immunofluorescence and Autophagy Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Differentiated EBs and monolayer cultures were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 at RT. The following primary antibodies were used: MF20 (1 : 50, Developmental Studies Hybridoma Bank); βIIITubulin (1 : 400, Sigma-Aldrich). After washing in 0.5% Tween-1 × PBS, cells were incubated with secondary antibodies (1 : 200, Alexa Fluor, Molecular Probes Inc., Life Technologies).
For autophagy detection, cytospin samples (see above) were stained with LysoTracker Red DND-99 dye. Briefly, cells (50 000 cells/spot) were washed with PBS 1 × , stained with LysoTracker Red DND-99 dye (Molecular Probes Inc.) for 1 min at room temperature. Cells were gently washed and fixed in 4% PFA for 30 min, washed three times with PBS, and stained with membrane stain WGA-Alexa Fluor 488 Conjugate (Invitrogen) following the manufacturer's instructions. Cell nuclei were counterstained with Hoechst 33342 (Invitrogen). Images were obtained using the DMI6000B microscope and the DFC 350FX B/W digital camera (Leica, Solms, Germany). Leica FW4000 and AF6000 software were used for image acquisition/elaboration. Confocal images were acquired at × 63 magnification on a LSM710 confocal fluorescence microscope (Carl Zeiss Inc., Jena, Germany) using the ZEN 2008 software (Carl Zeiss Inc.).
For autophagy detection, cytospin samples (see above) were stained with LysoTracker Red DND-99 dye. Briefly, cells (50 000 cells/spot) were washed with PBS 1 × , stained with LysoTracker Red DND-99 dye (Molecular Probes Inc.) for 1 min at room temperature. Cells were gently washed and fixed in 4% PFA for 30 min, washed three times with PBS, and stained with membrane stain WGA-Alexa Fluor 488 Conjugate (Invitrogen) following the manufacturer's instructions. Cell nuclei were counterstained with Hoechst 33342 (Invitrogen). Images were obtained using the DMI6000B microscope and the DFC 350FX B/W digital camera (Leica, Solms, Germany). Leica FW4000 and AF6000 software were used for image acquisition/elaboration. Confocal images were acquired at × 63 magnification on a LSM710 confocal fluorescence microscope (Carl Zeiss Inc., Jena, Germany) using the ZEN 2008 software (Carl Zeiss Inc.).
8
Quantifying Telomere Length Using FISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed telomere FISH16 using a peptide nucleic acid (PNA) probe specific to telomeres, and labeled with TelC‐Alexa488 (PNA Bio, Newbury Park, CA,) as per the manufacturer's instructions with modifications. Briefly, 0.2 µl of PNA probe was added to 20 µl of hybridization solution which was used to cover cells attached to slides. Hybridization was at 80°C for 10 min, then 37ºC overnight. After washing, slides were incubated with 10 μM ZnPP 2 h at room temperature, washed twice in PBS, and mounted with VECTASHIELD. Confocal fluorescence microscopy used a Zeiss LSM710 confocal fluorescence microscope using 63x oil objective.
9
Cryosectioning and Fluorescent Imaging of Locust Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The locusts were quickly frozen by liquid nitrogen and then embedded with O.C.T. compound (O.C.T. is abbreviation of Optimal Cutting Temperature, Sakura Finetek, USA). The embedded locusts were stored at −20°C overnight for cryosection. Next, 10 μm cryosections of different treatment locusts were prepared. These sections were stained with 2 μM DHE (Sigma, USA) for 1 h in a humidified box and then fixed in 4% (wt/vol) paraformaldehyde for half an hour. Finally, the samples were washed thrice with phosphate-buffered saline [42 (link)]. Hoechst 33342 (Invitrogen, USA) was used to stain the cell nucleus. The images were captured on an LSM 710 confocal fluorescence microscope (Zeiss, Germany).
10
Exosome-Bead Binding Assay for Tumor Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the exosome-bead binding assay, 1.5 μg/μL A549, H1975, or patient tumor-derived exosomes in 200 μL were added into 1.5 mL tube followed by 100 TentaGel beads coated with LXY30 or S-LXY30 at 37 °C for 60 min, respectively. The exosome-beads were then washed three times in PBS. After the wash, Alexa Fluor® 647 mouse anti-human CD63 antibody (Biolegend, San Diego, CA) was added into the tube, incubating for 1 h and then washed three times in PBS. Next, A549 exosome-bead and H1975 exosome-bead binding were visualized under a LSM710 confocal fluorescence microscope (Zeiss, Germany).
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!