IHC staining on paraffin-embedded sections was performed by a standard fluorescent IHC protocol, as previously described [28 (link)]. Sections were immunolabeled with the primary antibodies of monoclonal GFP (1:400, SCB) or rabbit polyclonal GFP (1:400, Cell Signaling, Danvers, MA), monoclonal β3-tubulin (TUBB3, 1:500, SCB), rabbit anti-glutamine synthetase (GS, 1:600, SCB), monoclonal proliferating cell nuclear antigen (PCNA, 1:200, SCB), rabbit anti Bcl-associated X protein (Bax, 1:100, SCB), rabbit polyclonal GDNF (1:400, SCB), monoclonal STRO-1 (1:200, Life Technologies), rabbit polyclonal glial fibrillary acidic protein (GFAP, 1:4000, Dako, Carpinteria, California), and α-smooth muscle actin (SM actin, Sigma-Aldrich, 1:1000), with BSA replacement of the first antibody as the negative control. The appropriate fluorophore-conjugated (Alexa 488 or Alexa 594) secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used to reveal the primary antibodies. The sections were examined and images captured using a Nikon TE2000-S fluorescence microscope (El Segundo, CA) with filters suitable for selectively detecting the green and red fluorescence, and a QuantiFire digital camera (Optronics, Santa Barbara, CA).
Te 2000s fluorescence microscope
Manufactured by Nikon
Sourced in United States, Japan
The Nikon TE-2000S is a fluorescence microscope designed for laboratory use. It is capable of providing high-resolution imaging of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Ultra view lci confocal imaging system, by Nikon (2 mentions)
Planapo360 immersion oil objective, by Nikon (2 mentions)
Alexa flour 488 goat anti mouse igg, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Avantor (2 mentions)
Sm actin, by Merck Group (1 mentions)
Fv1200, by Olympus (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Alexa fluor 647 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Alexa fluor 488 conjugated chicken anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Apo 60 oil immersion lens, by Nikon (1 mentions)
Tritc hyq, by Nikon (1 mentions)
Pegfp n1 plasmid, by Takara Bio (1 mentions)
Enhanced chemiluminescence reaction kit, by Cytiva (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
26 protocols using te 2000s fluorescence microscope
1
Fluorescent Immunohistochemistry Protocol for Paraffin Sections
2
Apoptosis and Nuclei Morphology Assay
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AO and Hoechst 33258 were used to test the morphological changes of nuclei and apoptosis, respectively. At 6 h, cells were stained with AO (5 μg/mL) at room temperature for 5 min or stained with Hoechst 33258 (10 μg/mL) for 15 min. After that, cells were observed under a laser scanning confocal microscopy (Olympus FV1200, Japan) or a TE2000S fluorescence microscope (Nikon, Japan), respectively.
3
Cytotoxicity Assay with 5-Fu and Curcumin
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Cells were mono-treated with 5-Fu or curcumin for 24 h or 48 h, and co-treated with 5-Fu and curcumin in different combinations basing on their concentrations and treatment time points. Cell proliferation was examined using the Cell Counting Kit-8 (CCK-8) (Beyotime), according to the manufacturer’s protocols. The cell morphology was observed using a TE2000-S fluorescence microscope (Nikon).
4
PAR-1 Receptor Cleavage Visualization
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HUVEC cells cultured on coverslips were stimulated with human MMP-9 for 5 minutes, as described above. The cells were then washed with HBSS without Ca2+ and Mg2+, fixed with 100% methanol, blocked with 5% BSA in PBS followed by incubation with anti-PAR-1 receptor antibody (ATAP-2, Zymed Laboratories Inc.; Bedminster, NJ). Anti-PAR-1 (ATAP-2) antibody recognizes intact and cleaved forms of PAR-1 receptor. Finally, cells were incubated with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Invitrogen; Waltham, MA). Cleavage of PAR-1 receptor was visualized using Perkin Elmer Ultra VIEW LCI confocal imaging system with Nikon TE2000-S fluorescence microscope and PlanApo360 immersion oil objective (numerical aperture 1.4) at room temperature. Ultra VIEW Imaging Suite software (version 5.5.0.4) was used for image processing.
5
Hoechst 33258 Staining for Apoptosis
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Hoechst 33258 staining was used to observe apoptotic morphology. At 48 h, cells in all groups were stained with 10 μg/mL Hoechst 33258 for 15 min. Cells were gently washed with PBS once. Nuclear condensation and fragmentation were observed under a TE2000S fluorescence microscope (Nikon, Japan).
6
Visualizing Golgi Positioning in Wound Cells
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As previously described (28 (link),29 (link)), the wounded cells were fixed with cold 4% paraformaldehyde at 4°C for 10 min and stained with the cis-Golgi matrix protein of 130 kDa (GM130) to visualize Golgi positioning after 16 h. A total of 7 µg/ml anti-GM130 antibody (cat. no. ab169276; Abcam) were incubated with cells at 4°C overnight. The appropriate secondary antibody conjugated with rhodamine were incubated for 1 h at room temperature. Then, 4′6-diamidino-2-phenyl-indole (DAPI) staining was performed as previously described (29 (link)). Cell images were acquired using a Nikon TE2000S fluorescence microscope (magnification, ×20) and were analyzed using ImageJ Fiji software (version 1.53g 4; National Institutes of Health). Cell orientation was determined only for cells at the wound edge. The cell was divided into three 120° regions, with one region facing the wound edge. The cell was recognized to possess an aligned Golgi only when its Golgi realigned to the 120° region facing the wound edge. The cell positioning angle was calculated between a line along the long axis of the nucleus and a line tracing the wound front. For example, cells aligned perpendicular to the leading edge demonstrated a nearly 90° orientation, whereas cells aligned parallel to the wound front had a 0° orientation. For each experiment, ≥20 cells were examined.
7
MMP-9 Activation of ERK Signaling in HUVEC
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HUVEC cells cultured on coverslips were incubated for 5 minutes with human MMP-9 (Calbiochem; San Diego, CA) at the concentration range of 0.1–0.01 nM in medium without serum containing sodium orthovanadate (1 mM) (Sigma Aldrich; St. Louis, MO). In some experiments HUVEC cells were treated with monoclonal antibodies against PAR1 receptor (WEDE15 and ATAP2; Immunotech Laboratories Inc.; Monrovia, CA and Santa Cruz Biotechnology; Dallas, TX, respectively) for 30 minutes at 4°C prior to incubation with human MMP-9. After 5 minutes of stimulation with human MMP-9, the cells were washed with HBSS without Ca2+ and Mg2+, fixed with 100% methanol, permeabilized with 0.5% (vol/vol) Triton X-100 in PBS, blocked in 5% BSA in PBS, and incubated overnight at 4°C with anti-pERK (Thr202/Tyr204) antibody (Cell Signaling Technology; Danvers, MA). To visualize phosphorylation of ERK, HUVEC cells were then incubated with Alexa Fluor 488 conjugated chicken anti-rabbit antibody (Invitrogen; Waltham, MA). The cells were observed using Perkin Elmer Ultra VIEW LCI confocal imaging system with Nikon TE2000-S fluorescence microscope and PlanApo360 immersion oil objective (numerical aperture [NA] 1.4) at room temperature. Ultra VIEW Imaging Suite software (version 5.5.0.4) was used for image processing.
8
Quantifying Vacuole Formation in FL5.12 Cells
9
Immunofluorescent Detection of HMGB1 and SIRT6
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To detect immunofluorescence labeled HMGB1 (red) and SIRT6 (green), tissue sections were first washed with PBS and Triton X-100 for 10 min. Sections were then blocked in 10% nonimmune serum for 1 h at room temperature and incubated in SIRT6 (1:100, 13572-1-AP; ProteinTech Group, Inc., Chicago, IL, USA) and HMGB1 (1:100, 66525-1-lg; ProteinTech Group, Inc.) primary antibodies overnight at 4 °C. Sections were then incubated with secondary antibodies for 2 h and then nuclei were counterstained with DAPI (blue) for 2 min and examined under a TE2000-S fluorescence microscope (Nikon, Tokyo, Japan). All antibodies for specific protein examples have been validated with western blot analysis (Supplementary Fig. 1 ).
10
Hoechst 33258 Staining for Apoptosis
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Hoechst 33258 staining was used to observe apoptotic morphology of MCF-7 cells treated with different components from CPWE. At 48 h, cells in all groups were stained with 10 μg/mL Hoechst 33258 for 15 min and then were gently washed with 1x PBS once. Nuclear condensation and fragmentation were observed under a TE2000S fluorescence microscope (Nikon, Japan).
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