Cells were transfected with an monomeric Red Fluorescent Protein-Green Fluorescent Protein-LC3 (mRFP-GFP-LC3) plasmid, and the images were captured by a fluorescence confocal microscope (Carl Zeiss, Dresden, Germany), using 63× oil objectives. GFP+/RFP+ LC3 puncta were yellow, and GFP−/RFP+ LC3 puncta were red. The average numbers of LC3 puncta per cell were determined on the basis of the data collected from >10 cells. The experiments were repeated three times.
Confocal fluorescence microscope
Manufactured by Zeiss
Sourced in Germany, United States
The Confocal fluorescence microscope is an optical imaging technique that allows for high-resolution, three-dimensional imaging of samples. It uses focused laser light to illuminate and excite fluorescent molecules within the sample, and a pinhole aperture to block out-of-focus light, resulting in sharp, high-contrast images.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Dapi 32670, by Merck Group (3 mentions)
Prolong gold antifade reagent with dapi, by Zeiss (3 mentions)
Anti α actinin, by Merck Group (3 mentions)
Vimentin, by Abcam (2 mentions)
Dapi staining solution, by Thermo Fisher Scientific (2 mentions)
Mitotracker red, by Thermo Fisher Scientific (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
α smooth muscle actin, by Merck Group (2 mentions)
α act, by Merck Group (2 mentions)
Gata4, by Santa Cruz Biotechnology (2 mentions)
Anti troponin t, by Merck Group (2 mentions)
Anti gata4, by Santa Cruz Biotechnology (2 mentions)
Live dead assay kit, by BestBio (2 mentions)
Pab1620, by Merck Group (1 mentions)
Pab240, by Merck Group (1 mentions)
Jc 1 fluorescent probe, by Beyotime (1 mentions)
β actin, by ABclonal (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
81 protocols using confocal fluorescence microscope
1
Monitoring Autophagy via mRFP-GFP-LC3
2
Immunofluorescence Detection of p53 Variants
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Cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min, incubated in 0.2% Triton X-100 for 5 min and in PBS with 1% BSA for 10 min, and stained overnight with primary antibody at 4 °C (wtP53 protein-specific antibody PAb 1620 and mutP53 antibody PAb 240, Merck Millipore, Boston, MA, USA). Then, separate staining with fluorescein-labeled secondary antibody (Alexa Fluor 555 for PAb 1620 and Alexa Fluor 488 for PAb 240, Invitrogen, Waltham, MA, USA) was performed for 1 h at room temperature, followed by three washes and staining with DAPI. The cells were finally washed and visualized under a fluorescence confocal microscope (Carl Zeiss, Oberkochen, Germany).
3
Quantifying Epithelial-Mesenchymal Transition
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2 × 104 cells/well 4T1 cells were inoculated in a 24-well culture plate with cell slides placed at the bottom in advance, so that the cells expand to 60–70% before experiment. The cells on the slides were washed by PBS for three times and then fixed with 500 μl 4% polyformaldehyde at room temperature for 15 min. After that, pre-cooled 0.2% Triton X-100 diluted in PBS was used to permeabilize the cells for 5 min. Then, the cells in each well were blocked with 500 μl goat serum containing 0.3% Triton X-100 at room temperature for an hour. After removal of the goat serum, the cells were covered with the mixed primary antibodies of E-cadherin (CST, Boston, Massachusetts, USA) and vimentin (Abcam, Cambridge, UK) overnight at 4 °C under dark conditions, and the two primary antibodies were coupled with Alexa Fluor® 594 and Alexa Fluor® 488, respectively. Afterwards, cells were washed three times using PBS, stained with DAPI staining solution (Invitrogen, Camarillo, CA, USA) for 10 min, washed, and sealed with BrightMount (Abcam, Cambridge, UK). Cells on the slides were observed by using a fluorescence confocal microscope (Zeiss, Oberkochen, Germany) with images subsequently obtained.
4
Immunofluorescence Staining of TSGH 8301 Cells
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TSGH 8301 cells seeded at 3 × 104 cells on coverslips
overnight were fixed in 4% paraformaldehyde on ice for 15 minutes,
washed 3 times with PBS, and then permeabilized with 0.1% Triton X-100
in PBS for 15 minutes at room temperature. The cells were further
blocked in 10% goat serum for 1 hour before the required primary
antibody was applied. After washing with PBS, the cells were incubated
with Alexa 488- or FITC-conjugated secondary antibody (BD
Biosciences). Cell nuclei were counterstained with DAPI 32670 (Sigma).
After extensive washing, coverslips were mounted onto glass microscope
slides, and cells were viewed with a fluorescence confocal microscope
(Zeiss, Jena, Germany).
overnight were fixed in 4% paraformaldehyde on ice for 15 minutes,
washed 3 times with PBS, and then permeabilized with 0.1% Triton X-100
in PBS for 15 minutes at room temperature. The cells were further
blocked in 10% goat serum for 1 hour before the required primary
antibody was applied. After washing with PBS, the cells were incubated
with Alexa 488- or FITC-conjugated secondary antibody (BD
Biosciences). Cell nuclei were counterstained with DAPI 32670 (Sigma).
After extensive washing, coverslips were mounted onto glass microscope
slides, and cells were viewed with a fluorescence confocal microscope
(Zeiss, Jena, Germany).
5
Mitochondrial Dysfunction Analysis in ZFL Cells
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Mitochondrial membrane potential of ZFL cells were measured by JC-1 fluorescent probe (Beyotime, China). After treatment, cells were incubated with JC-1 working solution at 28°C for 30 min. Subsequently, cells were washed with JC-1 buffer solution for two times. Then photographed with Zeiss fluorescence confocal microscope at proper fluorescence intensity (485 and 590 nm). Results were expressed as the ratio of the red/green fluorescence intensity, which represented the degree of mitochondrial damage.
6
Measuring Cellular Proliferation with EdU Assay
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The EdU assay was conducted in accordance with the manufacturer’s handbook (RiboBio, Guangzhou, China). After hRECs were seeded in a 48-well plate, 50 μM EdU was added to each well, and the plate was incubated at 37 ℃ for 4 h. The cells were fixed in 4% paraformaldehyde and permeabilized by 0.5% Triton X-100 then sequentially treated with Apollo 488 fluorescent dye solution and 4',6-diamidino-2-phenylindole (DAPI) solution. The image was captured using a fluorescence confocal microscope (Zeiss, Oberkochen, Germany).
7
Comprehensive Antibody Validation for Western Blotting and Immunofluorescence
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Primary antibodies were purchased from Abcam (Cambridge, MA, USA; HR, ab202840), Boster (Pleasanton, CA, USA; Hr, A04817), Aviva (Aviva Systems Biology, San Diego, CA, USA; CELF2, ARP40323), Sigma (St. Louis, MO, USA; FLAG, F1804; β-actin, A1978), and ABClonal (Woburn, MA, USA; H3K9me1, A2355; H3K9me2, A2359; CDK15, A13163; NR2F1, A16437; CA9, A1658). For Western blotting, approximately 30 µg of total cell lysate from each sample was loaded. IF staining was performed as described in Supplementary Materials and Methods [19 (link)]. IF images were acquired using a fluorescence confocal microscope (Zeiss, Thornwood, NY, USA). All IF experiments were performed in at least three independent experiments.
8
Immunofluorescence Staining Protocol
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Immunofluorescence staining was performed as previously reported17 (link).Briefly, cultured cells on glass coverslips or frozen tissue sections (8 μM thickness) were fixed in 4% paraformaldehyde for 10 min and blocked with blocking buffer (0.1% Triton X-100 and 10% normal serum in PBS) for 1 h. Fixed cells or tissue sections were then incubated with the indicated primary antibodies for 2 h followed by fluorochrome-conjugated secondary antibodies. After being washed with PBS, the slides were incubated with DAPI (Sigma) for nuclear staining. Images were acquired with a fluorescence confocal microscope (Zeiss, Thornwood, NY, USA).
9
In Situ DNA Fragmentation Detection
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For in situ detection of DNA fragmentation in paraffin-embedded tissue sections, the TUNEL method was performed using the In Situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland). Briefly, tumor sections were adhered to silane-coated slides and allowed to dry at 60 °C oven overnight. Subsequently, slides were deparaffinized and rehydrated. Protein digestion was done by incubating tissue in 20 μg/ml proteinase K (Worthington biochemical Co., Lakewood, NJ) for 15 min at room temperature. Antigen retrieval was done by immersing slides in 0.01 M citrate buffer, pH 6.0, in a microwave 750W for 1 min, followed by rapid cooling (30 min). Subsequently, the slides were immersed in Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum at room temperature for 30 min. TUNEL reaction mixture was added to slides and incubated at 37 °C in a humidified chamber for 1 hr. Tumor cell nuclei were stained with DAPI 32670 (Sigma Co., Steinheim, Germany). After extensive washing, the slides were mounted onto glass coverslips, and analyzed using an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm (green) by fluorescence confocal microscope (Zeiss, Jena, Germany).
10
Immunostaining and Quantification of Ly6G+ Cells
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Embedded colon sections were immunostained with antibodies (ZO1, Occludin and Ly6G), as previously described (Chung et al., 2014 ). Images were scanned using a fluorescence confocal microscope (Zeiss, Jena, Germany). Five fields of view from each sample were selected randomly, and Image‐Pro Plus software was used to calculate the Ly6G+ cell ratio.
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