Flat-mounted, PFA fixed, lamprey brains were dehydrated, embedded in paraffin, horizontally sectioned on a microtome (10 μm section thickness), and mounted on ColorFrost Plus slides. The samples were then processed for immunofluorescence as follows: rehydrated to PBS; incubated with Citrate Buffer (pH 6.0) for antigen retrieval (20 min boiling then 20 min RT); blocked with PBS with 0.1% bovine serum albumin and 0.2% Triton X-100 for 1 h RT; incubated with rabbit anti-p53 primary antibody diluted in blocking buffer overnight at 4°C (1:200; AbClonal, Cat A5761; RRID:AB_2766515 ); incubated with anti-rabbit AlexFluor+ 594 secondary antibody for 1 h RT in the dark (1:400; Invitrogen, Cat A32740; RRID:AB_2762824 ); and mounted under glass coverslips with Fluoromount-G (Southern Biotech, Birmingham, AL, USA). The samples were thoroughly washed with PBS between each step. Fluorescence images were captured via confocal microscopy (Nikon C2 microscope, 10× objective) and processed with NIS-Elements and Adobe Photoshop.
C2 microscope
Manufactured by Nikon
Sourced in Canada, Japan
The Nikon C2 microscope is a high-performance confocal imaging system. It features a compact and modular design, allowing for flexibility in configuration and application. The C2 provides advanced imaging capabilities, enabling users to capture high-resolution, three-dimensional images of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
A11034, by Thermo Fisher Scientific (2 mentions)
D9542, by Merck Group (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Plan apochromat 63, by Zeiss (1 mentions)
Ab108349, by Abcam (1 mentions)
A11032, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx plus reagent, by Thermo Fisher Scientific (1 mentions)
Anti γh2ax ser139 primary antibody, by Merck Group (1 mentions)
Transwell chamber, by Corning (1 mentions)
F 4500 fluorescence spectrofluorimeter, by Hitachi (1 mentions)
A1r confocal laser microscope system, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Comet assay kit, by Abcam (1 mentions)
C2si camera, by Nikon (1 mentions)
Elements image analysis software, by Nikon (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti γh2ax ser139, by Merck Group (1 mentions)
Ab176759, by Abcam (1 mentions)
34 protocols using c2 microscope
1
Immunostaining of Lamprey Brain Sections
2
Dendritic Spine Analysis in Mice
3
Confocal Laser Scanning Microscopy Protocols
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CLSM was either performed on a Nikon C2+ confocal laser scanning microscope or on a LSM 510 (Zeiss) confocal laser scanning microscope. The Nikon C2+ microscope was equipped with four lasers: a 405 nm diode laser (100 mW, Coherent Inc.), a 488 nm DPSS Laser (10 mW, Melles Griot GmbH), a 543 nm HeNe laser (5 mW, Melles Griot GmbH), and a 642 nm diode laser (45 mW, Melles Griot GmbH). The intensity of the laserlight was controlled by an acusto optical tunable filter (AOTF). We used a 60× violet corrected oil objective with a NA of 1.4 for imaging (Plan Apo VC 60× H, Nikon). The LSM 510 microscope was equipped with three lasers: a multiline argon laser (458 nm, 477 nm, 488 nm, 514 nm, 30 mW, LASOS Lasertechnik), a 543 nm HeNe laser (1 mW, LASOS Lasertechnik) and a 633 nm HeNe laser (5 mW, LASOS Lasertechnik). The intensity of the laser light was controlled by an acusto optical tunable filter (AOTF). For imaging a 63× objective with a NA of 1.4 (Plan Apochromat 63×, Zeiss) was used.
4
Immunostaining Protocol for Cell Imaging
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For immune staining, cells were grown on poly-D-lysine coated 8-well microscope slides (80826-IBI, Ibidi). Cells were fixed in 4% paraformaldehyde and stained (Ulrich et al., 2018 (link)) with primary antibodies: 0.168 μg/mL SUZ12, 0.842 μg/mL EZH2, 1.6 μg/mL H3K27me3 or 1.5 μg/mL P16 (ab108349, Abcam). Detection by fluorescent laser confocal microscopy (Nikon C2 microscope) was done with 2 μg/mL secondary antibodies anti-mouse IgG Alexa594, anti-rabbit IgG -Alexa 488 or anti-rabbit IgG-Alexa 647 (A-11032, A-11034, A21245, Thermo Fisher Scientific). Nuclei were counterstained with 0.5 μg/mL DAPI (D9542, Sigma-Aldrich). Images were acquired by sequential excitations (line-by-line scan) with the 405 nm laser (464/40 emission filter), the 488 nm laser (525/50 nm filter), the 561 nm laser (561/LP nm filter) and the 650 nm laser (594/633 emission filter). ImageJ was used for all image quantifications.
5
Polyglutamine Aggregation Dynamics
6
Visualizing Autophagy Dynamics via GFP-LC3
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Using lipofectamine LTX plus reagents (Thermo Fisher), cells were transfected with GFP-LC3 plasmid, gifted by Prof. Pu Xia, Fudan University, China. LC3 puncta were visualized under treatments. Immunofluorescent staining of γ-tubulin was performed in cells fixed with 4% paraformaldehyde, permeabilized in 2% Triton X-100, blocked in 3% fatty acid-free bovine serum albumin (BSA), incubated with fluorophore-bound γ-tubulin antibody (Thermo Fisher, #MA1-850-A555) and counterstained in ProLong™ Gold Antifade Mountant with DAPI. Confocal microscopy was carried out using a Nikon C2 microscope.
7
Quantifying DNA Damage in PBMCs
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PBMCs were fixed with 1% formaldehyde fixation solution in phosphate-buffered saline (PBS) for 10 min at room temperature. Cells were rinsed three times with PBS and incubated with 0.1% Triton X-100 in PBS for 5 min and blocked with 2% BSA in PBS for 30 min. Cells were immunostained with anti-γH2AX (ser139) primary antibody (Merck) (1:3000, diluted in 0.2% BSA in PBS) at 4 °C overnight. After this incubation period, cells were rinsed three times with PBS and incubated with Alexa Fluor® 488 anti-mouse secondary antibody (1:400, diluted in 0.2% BSA in PBS) for 1 h at room temperature. Cells were rinsed three times with PBS, and the coverslips were mounted in DAKO mounting medium on glass slides.
Confocal image stacks were captured with a Nikon C2+ microscope, using a 20× objective. Between 100 and 120 cells per patient were acquired. ImageJ analysis software was used to generate the zeta projections from 10 to 15 stacks (0.8 mm thickness each). Quantitative analysis of immunofluorescence data was carried out with a histogram analysis of the fluorescence intensity at each pixel across the nucleus. The data were normalized by the number of nuclei and are expressed in arbitrary fluorescence units (AU).
Confocal image stacks were captured with a Nikon C2+ microscope, using a 20× objective. Between 100 and 120 cells per patient were acquired. ImageJ analysis software was used to generate the zeta projections from 10 to 15 stacks (0.8 mm thickness each). Quantitative analysis of immunofluorescence data was carried out with a histogram analysis of the fluorescence intensity at each pixel across the nucleus. The data were normalized by the number of nuclei and are expressed in arbitrary fluorescence units (AU).
8
Automated Synaptic Puncta Quantification
9
Evaluating Anti-angiogenic Effects of 6h
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The 96-well plate was coated with 50 μl of Matrigel mixed with an equal volume of FBS-free medium, which was allowed to polymerize at 37 °C for 30 min. A549 cells or H460 cells were treated with various concentrations of 6h for 24 h, and the CM from each group was collected. After being pretreated with ECM or ECM containing 50% CM for 24 h, HMEC-1 cells were inoculated on the Matrigel. After 6 h of incubation, the formation of tubes was observed using a Nikon C2 microscope.
10
Apoptosis Evaluation by Hoechst 33258 and Annexin V
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The morphological changes of apoptotic cell nuclei were evaluated by Hoechst 33258 staining. Apoptosis assay was performed as described previously (40 (link)). Cells were processed according to the operating instructions of the annexin V–FITC/PI staining kit. The samples were detected by a Nikon C2 microscope or FACS. Apoptosis rate (%) = early apoptosis rate (PI−; annexin V–FITC+) + late apoptosis rate (PI+; annexin V–FITC+).
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