The brain sections and neural stem cells used in the immunofluorescent staining were utilized for the detection of damaged cells in TUNEL assay using a Fluorescein In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). After washing with PBS, the sections and cells were incubated for 10 min with pre-cold ethanol-acetic solution (3:1), followed by incubation with 5% Triton-X 100 (Sigma). Subsequently, the sections and cells were incubated 90 min with TdT-enzyme buffer supplemented with fluorescein-dUTP, followed by Hoechst 33258 (Invitrogen, Germany). The signals were detected by a laser confocal fluorescence microscopy (Leica, Germany).
Laser confocal fluorescence microscopy
Manufactured by Leica
Sourced in Germany
Laser confocal fluorescence microscopy is an imaging technique that uses a laser to excite fluorescent molecules within a sample. The emitted fluorescence is detected and reconstructed into a high-resolution image. This method provides optical sectioning, allowing for the visualization of specific layers within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluorescein in situ cell death detection kit, by Roche (1 mentions)
20 mm glass bottom dish, by NEST Biotechnology (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab6142, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
6 protocols using laser confocal fluorescence microscopy
1
TUNEL Assay for Detecting Cell Death
2
Evaluating BMSC Proliferation Dynamics
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To study the proliferative ability of the BMSCs, cell growth was evaluated by manually counting at each passage. Briefly, 3 × 105 BMSCs were seeded in the 60 mm2 dish and passaged once the culture reached 80–90% confluence. Cell numbers were recorded and cells were replated at the same density each time. Cell proliferation was also examined by EdU assay. In brief, 1.5 × 105 BMSCs were plated in 20 mm glass bottom dish (NEST Biotechnology, China) and cultured overnight. After incubation with EdU for another 36 h, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed twice with 3% BSA in PBS, incubated with 0.5% Triton X-100 in PBS for 20 min and stained with the Click-iT EdU Imaging Kit (Life Technologies) according to the manufacturer’s instruction. Nuclear staining was performed with 4′, 6-diamidino-2-phenylindole (DAPI). Photomicrographs were taken by Laser confocal fluorescence microscopy (Leica, Germany). The percentage of proliferating cells was calculated based on counting EdU-positive cells/total cells in five individual visual fields.
3
Aptamer and Antibody Binding Assay
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Cells
were incubated with Cy5-labeled aptamers at a concentration of 100
nM and FITC-labeled anti-ITGB3 antibodies at 4 °C in the dark
for 30 min. After three washes with an ice-cold washing buffer, fluorescence
images were obtained using laser confocal fluorescence microscopy
(Leica, Germany).
were incubated with Cy5-labeled aptamers at a concentration of 100
nM and FITC-labeled anti-ITGB3 antibodies at 4 °C in the dark
for 30 min. After three washes with an ice-cold washing buffer, fluorescence
images were obtained using laser confocal fluorescence microscopy
(Leica, Germany).
4
Acridine Orange Staining for Cell Imaging
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The transfected cells (2 × 105 cells/well) were inoculated in a confocal culture dish until the cells’ adherence to the wall. Next, cells were incubated with acridine orange with a final concentration of 1 mg/L for 10 min, and they were observed and quantified by laser confocal fluorescence microscopy at excitation wavelength 488 nm and emission wavelength 510 nm (Leica, Mannheim, Germany).
5
Immunofluorescent Staining of TAOK1 in Brain and Neural Stem Cells
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For TAOK1 immunofluorescent staining of brain slice, after fixing the brain slice in 4% formalin overnight, and dehydrated in 10, 20 and 30% sucrose solutions for 3 days, the brain area containing SVZ was cut into 35-μm sections. For TAOK1 immunofluorescent staining of neural stem cells, the neural stem cells were collected and washed twice with PBS, and then fixed in 4% formalin overnight. After blocking with 10% donkey serum for 2 h, the brain sections and fixed neural stem cells were initially incubated with primary antibodies against TAOK1 (Rabbit, 1:500, ab150519, Abcam) for overnight at 4°C. After washing with PBS for three times (10 min/time), the brain slices and neural stem cells were stained with anti-rabbit secondary antibody-546 for 2 h, and incubated in DAPI for 10 min. The signals were detected with a laser confocal fluorescence microscopy (Leica, Germany). The neural stem cells were additionally stained with Nestin (Mouse, ab6142, 1:1000, Abcam), GFAP (Rabbit, ab7260, 1:1000, Abcam), and Tuj1 (Rabbit, ab18207, 1:1000, Abcam) as described above.
6
Evaluation of p65 Translocation in FLS
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To evaluate the translocation of p65, ICC assay was performed according to the protocol. FLS in the anti-CD147 group were pretreated with 10 μg/mL anti-CD147 monoantibody for 4 hours before coculture with IL-1α-induced cartilage and continually given anti-CD147 treatment in two days of coculture setting. Cocultured with pretreated articular cartilage, cocultured FLS were fixed in ice-cold 4% formaldehyde, permeabilized using 0.1% Triton X-100. Next, fixed cells were blocked with 5% BSA and incubated with primary antibody (anti-p65, CST) at 4°C for 16 hours. Furthermore, FLS were incubated with fluorescent secondary antibody (Anti-rabbit IgG (H+L), Alexa Fluor® 594 Conjugate, CST) and counterstained with DAPI. Laser confocal fluorescence microscopy (Leica) was used to detect fluorescent signals from FLS.
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