Seriated sections were treated with 10% H2O2 (Merck, Darmstadt, Germany) in PBS at pH 7.2 for 30 min to allow antibody-tissue interaction. After washing the slides with PBS, they were incubated with anti-tubulin - anti rabbit- (Sigma-Aldrich, St. Louis, MO, USA), anti-myosin -anti rabbit- (Sigma-Aldrich), or anti-active caspase-3 -anti rabbit-(Sigma-Aldrich) at a 1:100 dilution in PBS for 18 h at 4°C. Negative controls were made by omitting the primary antibody. Next, the slides were washed with PBS and incubated for 1 h in darkness at room temperature with secondary antibody anti-rabbit immunoglobulin coupled to FITC (Jackson ImmunoResearch, West Grove, PA, USA). Sections were counterstained with DAPI 10 g/mL (Sigma-Aldrich). Immunoassays were evaluated under a Nikon Eclipse E600 Fluorescence Microscope equipped with a Nikon Digital DXM1200F high-resolution color digital camera with a resolution of up to 12 million pixels. The objectives used were 40X and 100X. The slides were observed using DAPI (EX 330-380, BA 455-485), Texas red and Alexafluor 594 (EX 540-580, BA 600-660), and FITC filters (EX 465-495, BA 515-555). The images were recorded and processed with the Adobe Photoshop CS5 program.
Eclipse e600 fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Nikon Eclipse E600 is a fluorescence microscope designed for scientific and research applications. It features high-intensity illumination and advanced optics to enable detailed observation and analysis of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Axiovert 200m inverted microscope, by Zeiss (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Alexa 488 tyramide signal amplification, by Thermo Fisher Scientific (2 mentions)
Anti dig pod antibody, by Roche (2 mentions)
Dxm1200f digital, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti γh2ax antibody, by Merck Group (1 mentions)
Anti insulin and anti glucagon antibodies, by Abcam (1 mentions)
Senescence detection kit, by Abcam (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Anti rabbit hrp dab cell tissue staining kit, by R&D Systems (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Plan neofluar objective, by Zeiss (1 mentions)
Nis elements software, by Nikon (1 mentions)
Alexa 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Immu mount, by Thermo Fisher Scientific (1 mentions)
25 protocols using eclipse e600 fluorescence microscope
1
Immunohistochemistry of Cytoskeletal Proteins
2
Immunohistochemistry and Senescence Analysis of Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen tissues were sectioned and slides prepared by Histoserv, Inc. (Germantwon, MD, USA). Immunohistochemisty of γH2AX was performed following the instruction of the anti‐rabbit HRP‐DAB Cell & Tissue Staining Kit (R&D systems) with an anti‐γH2AX antibody (1:100; Sigma‐Aldrich). For insulin and glucagon staining, slides were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X‐100 in PBS, blocked in a solution containing 5% normal donkey serum, 1% bovine serum albumin, 0.2%, gelatin and 0.2% Triton X‐100 in PBS, hybridized with anti‐insulin and anti‐glucagon antibodies together (1:500; Abcam, Cambridge, MA, USA) for 16 h at 4 °C, washed twice in PBS with 0.2% Triton X‐100 for 30 min and once in PBS for 30 min, hybridized with secondary antibodies (1:200, anti‐mouse Alexa Fluor 488 and anti‐rabbit Alexa Fluor 568; Invitrogen, Carlsbad, CA, USA), and then washed as described above. Finally, the slides were mounted with ProLong Gold Antifade reagent containing DAPI (Invitrogen). The expression of senescence‐associated β‐galactosidase was determined following the instruction of a Senescence Detection Kit (BioVision, San Francisco, CA, USA). Images were visualized and captured under a Nikon Eclipse E600 fluorescence microscope (Nikon Inc., Melville, NY, USA).
3
Osteoclast Cytoskeleton and Nuclei Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After culturing the cells were fixed with 4 % PFA in PBS. The actin cytoskeleton was stained with Alexa 488-conjugated phalloidin (200 U/ml stock diluted 1:100 in PBS; Invitrogen Europe, Paisley, UK) for 20 minutes at +37 °C. Nuclei were stained with Hoechst 33258 (1 mg/ml stock diluted 1:800 in PBS; Sigma-Aldrich) for 10 minutes at room temperature. Staining for osteoclast-specific enzyme TRACP was carried out with a commercial acid phosphatase leukocyte kit (Sigma-Aldrich) for 20 minutes at +37 °C. The samples were mounted in 70 % glycerol-PBS and viewed in a Nikon Eclipse E600 fluorescence microscope (Tokyo, Japan) and Plan 10 × objective. Multinuclear cells with three or more nuclei were counted from each bone slice from five randomly chosen microscope fields, bone slice n ≥ 3. The number of nuclei were counted from 4 multinuclear cells from 5 randomly chosen areas. Images were taken with QImaging MicroPublisher 5.0 RTV camera and QCapture 2.90.1 software (QImaging, Surrey, Canada). Confocal images were taken with LSM 510 META confocal microscope combined with an Axiovert 200 M inverted microscope (Carl Zeiss, Oberkochen, Germany) with 20 x Plan Neofluar objective (Carl Zeiss).
4
Immunofluorescence Staining of Cultured Neurons
5
Staining and Visualization of Actin Cytoskeleton and Osteoclasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The actin cytoskeleton of the cells was stained with Alexa 488-conjugated phalloidin (200 U/ml stock diluted 1:100 in PBS; Invitrogen Europe, Paisley, UK) for 20 minutes at +37 °C. The nuclei were stained with Hoechst 33258 (1 mg/ml stock diluted 1:800 in PBS; Sigma-Aldrich) for 10 minutes at room temperature. The osteoclast-specific enzyme tartrate resistant acidic phosphatase (TRACP) was stained with a commercial acid phosphatase leukocyte kit (Sigma-Aldrich) for 20 minutes at +37 °C. The samples were mounted in 70% glycerol-PBS and viewed in a Nikon Eclipse E600 fluorescence microscope with Plan 10x/0.25 objective (Tokyo, Japan). Multinuclear cells with three or more nuclei were counted. Images were taken with the Nikon Eclipse E600 fluorescence microscope and Plan 20x/0.5 objective with QImaging MicroPublisher 5.0 RTV camera and QCapture 2.90.1 software (QImaging, Surrey, Canada). For demonstration of the number of stromal cells in the two culture types the cells were grown on glass slides and stained with Alexa 488-conjugated phalloidin as described earlier. Samples were mounted in Immu-Mount (Thermo Fisher Scientific, Waltham, MA) and viewed in an LSM 510 META confocal microscope combined with an Axiovert 200 M inverted microscope (Carl Zeiss, Oberkochen, Germany) with Plan Neofluar 10x/0.5 objective (Carl Zeiss).
6
Imaging and Quantification of Neuropeptide-Containing Dorsal Root Ganglion Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Specimens were analyzed in a Bio-Rad Radiance Plus confocal scanning microscope (Bio-Rad, Hemel, Hempstead, UK) installed on a Nikon Eclipse E 600 fluorescence microscope (Nikon, Tokyo, Japan) and, in some experiments, an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany) as in previous work [122 (link)]. The percentage of +NPs in DRGs was obtained as described [115 (link)]. Four to 8 sections of each DRG from 5 animals in each group were included in the analysis. The size distribution of +NPs with a visible nucleus was measured using the Nikon Eclipse E 600 fluorescence microscope with Wasabi Image Software. The +NPs were divided into small, medium-sized and large according to earlier studies [53 (link)]. The relative fluorescence levels (intensity) of sst2A-like immunoreactivity (LI) in DRGs before and after nerve injury were measured as described [115 (link)] using a Sarastro 1000 confocal laser-scanning system (Molecular Dynamics, Sunnyvale, Calif., USA).
7
Actin Filament Analysis in PC3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence microscopic analysis was performed to assess the actin filaments in PC3 cells without and with C12-HSL treatment using Acti-stain™ 488 fluorescent phalloidin (Cytoskeleton Inc., Denver, CO). Cells were cultured in chamber slides with DMSO vehicle (<0.01%) or with C12-HSL (50 μmol/L) in DMSO for 1 h, at 37°C and 5% CO2. Subsequently, the cells were then fixed with cold paraformaldehyde followed by addition of Triton-X 100 (0.1% in 1x PBS) to enhance membrane permeability. Cells were washed with phosphate buffered saline (PBS), labeled with phalloidin, and incubated for 1 h at RT. After washing with 1x PBS, cell nuclei were counterstained using 4′,6′-diamino-2-phenylindole (DAPI). The slides were visualized using a Nikon Eclipse E600 fluorescence microscope (Nikon Instruments Inc., Melville, NY).
8
Immunofluorescent Localization of FAM83H and MYC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the localization of FAM83H and MYC, immunofluorescence staining was performed. The HLE and HepG2 cells were fixed with methanol and incubated with anti-FAM83H (1:100, Bethyl Laboratories) and anti-MYC (1:100, Abcam) antibodies and then incubated with Alexa FluorVR 488 anti-rabbit IgG or Alexa FluorVR 594 anti-mouse IgG (Invitrogen, Carlsbad, CA). The slides were counterstained with DAPI, and the images were acquired using a Nikon ECLIPSE E600 fluorescence microscope (Nikon, Japan) and software (Nikon ACT-1 2.62, Nikon).
9
Immunofluorescent Detection of CXCR4 in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in chamber slides and then were fixed in 4% paraformaldehyde in PBS for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked for 1 hour at room temperature in PBS containing 5% goat serum (Invitrogen, Rockville, MD). Samples were then incubated in blocking buffer containing mouse monoclonal anti-human CXCR4 antibody or isotype antibody (R&D Systems) for 2 hours at room temperature and washed three times with PBS for 15 minutes. Cells were then incubated with secondary anti-mouse antibody conjugated with FITC (1 : 1000, Molecular Probes, Eugene, OR; http://www.lifetechnologies.com/ ) for 1 hour at room temperature. The samples were washed as above and mounted with 6-diamidino-2-phenylindole (DAPI; DAKO, Carpinteria, CA; http://www.dako.com/ ) containing mounting solution. The cells were examined under a Nikon Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan; http://www.nikon.com/ ).
10
Emodin Modulates Macrophage Migration
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!