ESCC cell lines and HEEpiC cells were grown on glass coverslips (NEST Biotechnology, China) to 70% confluence and then fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the rat monoclonal anti-ESE3 antibody at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson, USA) for 45 min at 37°C. The cells were rinsed in PBS between incubation steps and mounted with medium containing 4'-6-diamidino-2-phenylindole. Both fixed and living cells transfected with an enhanced green fluorescent protein (EGFP) expression plasmid were examined under an IX51 fluorescence microscope (Olympus, Japan).
Glass coverslips
Manufactured by NEST Biotechnology
Sourced in China
Glass coverslips are thin, transparent glass sheets used to cover and protect samples during microscopic examination. They provide a flat, smooth surface for mounting specimens and facilitate the use of various microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ix51 fluorescence microscope, by Olympus (1 mentions)
Phrodo green, by Thermo Fisher Scientific (1 mentions)
24 well culture plate, by Corning (1 mentions)
Ad gfp p62, by Beyotime (1 mentions)
Ad mcherry gfp lc3b, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Tcs sp2 aobs, by Leica (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Lsm 780 confocal laser scanning system, by Zeiss (1 mentions)
24 well polystyrene culture plates, by NEST Biotechnology (1 mentions)
Ab150131, by Abcam (1 mentions)
Ab75472, by Abcam (1 mentions)
Sc 7312, by Santa Cruz Biotechnology (1 mentions)
Af 4519, by R&D Systems (1 mentions)
Ab150073, by Abcam (1 mentions)
12 protocols using glass coverslips
1
Fluorescence Microscopy of ESE3 Expression
2
Mammalian Cell-Bacteria Coculture Assay
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Mammalian cells were seeded at 1 × 105 cells per well onto glass coverslips (14 mm, NEST) in 24‐well culture plates (Corning) to form monolayers. Then bacterial colonies were scraped and resuspended in PBS (0.01 m , pH = 7.2, Gibco) to pre‐incubate with pHrodo‐Green (Molecular Probes), except B. cereus pGFP 4412 expressing GFP. Finally, 4 × 106 colony‐forming units (CFUs) bacteria were cocultured with mammalian cells.
5‐week‐old female ICR mice (n ≥ 5) were infected intragastrically with 200 µL bacteria (B. cereus NVH0075/95 and E. coli ATCC25922) in 0.9% saline solution at 1 × 109 CFUs per mouse for 24 h. Meanwhile, the mice solely treated with saline solution were as the no antibiotic control. While infected mice were treated with 0.5 µg mL−1 ciprofloxacin and 2 µg mL−1 tetracycline were served as antibiotic treatments. More experimental details could be found in the supporting information.
5‐week‐old female ICR mice (n ≥ 5) were infected intragastrically with 200 µL bacteria (B. cereus NVH0075/95 and E. coli ATCC25922) in 0.9% saline solution at 1 × 109 CFUs per mouse for 24 h. Meanwhile, the mice solely treated with saline solution were as the no antibiotic control. While infected mice were treated with 0.5 µg mL−1 ciprofloxacin and 2 µg mL−1 tetracycline were served as antibiotic treatments. More experimental details could be found in the supporting information.
3
Monitoring Autophagy in Bacterial Infections
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To track autophagy, IEC‐6 cells were seeded on glass coverslips (14 mm, NEST) in a 24‐well plate and transfected with modified adenoviruses (Ad‐mcherry‐GFP‐LC3B and Ad‐GFP‐p62, Beyotime) at 2 × 106 plaque forming units (PFUs), according to the product's instruction. Then, labeled cells were cultured in DMEM with 10% FBS in the presence of B. cereus NVH0075/95 or E. coli ATCC29522, under the treatments of either ciprofloxacin or tetracycline (0.5 µg mL−1 ciprofloxacin and 4 µg mL−1 tetracycline for B. cereus; 0.25 µg mL−1 ciprofloxacin and 0.5 µg mL−1 tetracycline for E. coli) for 8 h. Lastly, coverslips were collected and fixed for confocal microscope analysis. LC3B was shown as green or red or the both, whereas the protein of p62 was shown in green.
4
Immunofluorescence Staining of Viral Proteins
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IPI-2I cells, BHK-21 cells, or HeLa cells in 24-well plates were seeded on glass coverslips (NEST). Cells were washed three times with phosphate-buffered saline (PBS) and then sealed with 4% paraformaldehyde in methanol for 15 min and methanol for 15 min. Bovine serum albumin (5%) diluted in PBS was used to block cells for 1 h, and mouse monoclonal antibody against PDCoV S, TGEV M, or anti-Flag rabbit polyclonal antibody was then added and incubated for 1 h. After three washes with PBS, Alexa Fluor-conjugated secondary antibodies were added and incubated for 1 h, followed by 0.01% 4′,6-diamidino-2-phenylindole (DAPI) staining for 15 min to detect nuclei. After three washes with PBS, fluorescent images were examined by confocal laser scanning microscopy (LSM 510 Meta, Carl Zeiss).
5
Immunofluorescence Staining of NONO Protein
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ESCC cell lines were grown on glass coverslips (Nest Biotechnology Co., Ltd., Wuxi, China) to 50% confluence and then fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized with 0.3% Triton X-100 for 20 min, blocked with 5% bovine serum albumin for 30 min, and then incubated with the mouse monoclonal anti-NONO antibody (Becton Dickinson) at 4°C overnight, followed by an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 45 min at 37°C. The cells were rinsed in PBS between incubation steps and mounted with medium containing 4′-6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were acquired using a fluorescence microscope (Olympus).
6
UBE2T Expression Regulation Analysis
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Cells with altered expression of UBE2T were cultured on glass coverslips (NEST, 801007) in 24-well plate and fixed by 4% paraformaldehyde for 15 min at 50–60% density followed by washing in PBS. After blocking in goat serum (1:10 in PBS) for 30 min, the coverslips were incubated with primary antibodies (diluted in primary stain diluting buffer, Beyotime, P0103) overnight at 4°C and secondary antibodies 1 hour at 37°C. Nuclei were visualized by 4,6-diamidine-2-phenylindole staining (DAPI, Solarbio, D8200). The coverslips touched face down a drop of Anti-fade Mounting Medium (Beyotime, P0126) on a slide and the fluorescence was captured by laser scanning confocal microscopy.
7
Immunofluorescence Imaging of Adherent Cells
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Adherent cells on glass coverslips (NEST Biotechnology, Wuxi, China) or cell suspensions collected by centrifugation were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Samples were blocked in 5% goat serum, stained with the appropriate fluorescence-coupled primary and secondary antibodies, and imaged by confocal microscopy (TCS SP2AOBS, Leica Microsystems, Wetzlar, Germany).
8
Cytoskeleton Visualization Protocol
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For cytoskeleton staining, cells were cultured on the glass coverslips (NEST), fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (Beyotime), and stained with Phalloidin (Sigma-Aldrich) according to the protocol provided by the manufacturer. Subsequently, 4ʹ,6-diamidino-2-phenylindole (Solarbio) was used to counterstain the cell nucleus. After mounting on the coverslips, the cells were visualized using a fluorescence microscope (Leica, Solms, Germany).
9
Immunofluorescence Staining of Cell Lines and Tumors
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S26 cells and cells expressing LMP1 or LMP2A, were grown on glass coverslips (NEST) for 48 hours. Cells were fixed by using 4% paraformaldehyde for 10 min, permeabilized in 0.1% Triton X-100 for 30 min, and blocked in 1% BSA for 1 hour. Then the cells were incubated with anti-Vimentin, anti-ZEB1, or anti-E-cadherin antibodies for 2 hours at room temperature. Fluor Alexa-555 conjugated anti-rabbit IgG and Fluor Alexa-555 conjugated anti-mouse IgG was used as secondary antibody.
For immunofluorescent staining of paraffin-embedded tumors, sections were deparaffinized and rehydrated, followed by heat-mediated antigen retrieval. Slides were permeabilized in 0.1% Triton X-100 for 30 min, blocked in 1% BSA for 1 hour, and then incubated with CD44 PE (1:100 dilution) and CD104 eFluor 660 (1:50 dilution) antibodies at 4°C overnight. Slides were visualized using a Zeiss LSM780 confocal laser scanning system.
For immunofluorescent staining of paraffin-embedded tumors, sections were deparaffinized and rehydrated, followed by heat-mediated antigen retrieval. Slides were permeabilized in 0.1% Triton X-100 for 30 min, blocked in 1% BSA for 1 hour, and then incubated with CD44 PE (1:100 dilution) and CD104 eFluor 660 (1:50 dilution) antibodies at 4°C overnight. Slides were visualized using a Zeiss LSM780 confocal laser scanning system.
10
MRSA Biofilm Transformation with GO-PEI-AsyycG
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A single colony of MRSA was selected from tryptose soya agar (TSA) plates and cultured in
liquid TSB medium to an OD600 value of 0.5, representing mid-exponential
growth. Two hundred and fifty microliters of the mid-exponential phase MRSA cultures were
placed in 24-well polystyrene culture plates (Nest Biotechnology, Wuxi, Jiangsu, China),
and a biofilm was established on glass cover slips (14 mm in diameter) (Nest
Biotechnology, Wuxi, Jiangsu, China) by culturing for 24 hr. The biofilms were then washed
three times with PBS buffer to remove planktonic bacteria before the addition of
transformation reagents. For the GO group, 2 µl of GO solution (with a
concentration of 50 mg/ml determined by cell viability assay) was added
to 24 hr old MRSA biofilms. For the ASyycG group, 2 µlof recombinant pDL278 ASyycG plasmid was added to the MRSA biofilm
according to our previous methods. For GO-PEI-ASyycG transformations, 2
µl of the GO-PEI-ASyycG solution, which already
included 200 ng of ASyycG plasmids, was added to 24 hr
MRSA biofilms. After intervention, all the MRSA biofilms were cultured at 37°C containing
5% CO2 for an additional 24 hr and prior to further investigation [32 (link)].
liquid TSB medium to an OD600 value of 0.5, representing mid-exponential
growth. Two hundred and fifty microliters of the mid-exponential phase MRSA cultures were
placed in 24-well polystyrene culture plates (Nest Biotechnology, Wuxi, Jiangsu, China),
and a biofilm was established on glass cover slips (14 mm in diameter) (Nest
Biotechnology, Wuxi, Jiangsu, China) by culturing for 24 hr. The biofilms were then washed
three times with PBS buffer to remove planktonic bacteria before the addition of
transformation reagents. For the GO group, 2 µl of GO solution (with a
concentration of 50 mg/ml determined by cell viability assay) was added
to 24 hr old MRSA biofilms. For the ASyycG group, 2 µlof recombinant pDL278 ASyycG plasmid was added to the MRSA biofilm
according to our previous methods. For GO-PEI-ASyycG transformations, 2
µl of the GO-PEI-ASyycG solution, which already
included 200 ng of ASyycG plasmids, was added to 24 hr
MRSA biofilms. After intervention, all the MRSA biofilms were cultured at 37°C containing
5% CO2 for an additional 24 hr and prior to further investigation [32 (link)].
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