The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were washed with PBS three times and then permeabilized with 0.3% Triton X-100 for 20 min. Furthermore, the cells were blocked in 5% ordinary goat serum for 1–2 h at room temperature. Rabbit antikeratin 14 and PAS antibodies (Thermo, Rockford, USA), and rabbit anti-P. aeruginosa (home-made antibody; 1:200) were added to the cells and incubated overnight at 4°C. After removing the primary antibody, the cells were washed three times using PBS. Alexa Fluor 594-labeled antibody (green) and Alexa Fluor 594-labeled antibody (red; Jackson Immuno Research Laboratories, West Grove, PA, USA; 1:500) were added to the cells as fluorescent secondary antibodies to detect primary antibodies. The fluorescent secondary antibodies were removed and washed three times with PBS, and the membrane was fixed on a glass slide by Vectashield Mounting Medium (H-1200, Vector Laboratories, Burlingame, CA) with 4’ 6-diamidino-2-phenylindole (DAPI). Colocalization images were acquired by the Leica TCS SP2 A0BS confocal system and analyzed by the Leica Confocal Software v. 2.6.1 (Leica, Germany).
Tcs sp2 a0bs confocal system
Manufactured by Leica
Sourced in Germany
The TCS SP2 A0BS Confocal System is a scientific laboratory instrument designed for high-resolution imaging and analysis of biological samples. It utilizes confocal laser scanning microscopy technology to capture detailed, three-dimensional images of specimens. The system is equipped with a range of features and capabilities to support advanced research and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Confocal software v 2, by Leica (4 mentions)
Vectashield mounting medium h 1200, by Vector Laboratories (1 mentions)
Confocal software version 2, by Leica (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Vectashield mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
6 protocols using tcs sp2 a0bs confocal system
1
Immunofluorescence Staining of P. aeruginosa
2
Confocal Laser Scanning Microscopy Biofilm Assay
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Concentrations of ERY, CIP, and TASA used in this assay were 500 μg/mL, 500 μg/L, and 20 mg/mL, respectively. For CLSM analysis of biofilm, above treated bacteria cultured on glass cover slides were incubated with 5 μL DNA fluorescent staining solution (the fluorescent staining solution was mixed by SYTO9 and propidium iodide prior to be applied). The plate was incubated at 37°C for 15 min in dark. After washing for 3 times with PBS, the aseptic cover glass was placed on a glass slide [20 (link)]. The stained biofilm was then examined with CLSM with parameters of green exciting light at 488 nm, red exciting light at 543 nm, 40x objective, and 10x eyepiece using a Leica TCS SP2 A0BS Confocal System and processed on Leica Confocal Software v.2.6.1 (Leica, Germany). Each group was repeated three times and 3 different fields were randomly selected for each sample. Then the selected fields were scanned layer by layer from 4 to 20 layers based on the thickness of biofilm. Viable bacteria were displayed in green fluorescent, dead bacteria were imaged in red fluorescent, and an area with both viable and dead bacteria was superimposed as aurantiacus fluorescence in images of CLSM.
3
Immunofluorescence Analysis of CFTR Expression
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The A549 cells cultured in collagen-coated cover slides were transfected with
pacAd5.huCFTR-ECL4HA plasmid for 12 hours and cultured in the presence or absence of
nicotine for additional 24 hours. The cells were then fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) at room temperature for 15 minutes, washed in PBS for 3× 5
minutes, and permeabilized with 0.3% Triton X-100 for 10 minutes at room temperature.
Nonspecific antibody binding was blocked using 5% normal donkey serum in PBS for 1 hour at
room temperature, after which primary antibodies of mouse anti-CFTR and rabbit anti-HA
were applied at a 1:100 dilution in PBS and applied to probe proteins of interest by
incubating slides at 4°C overnight. The primary antibody binding was detected using the
Alexa Fluor 488-labeled donkey-anti-mouse immunoglobulin G (IgG) secondary antibody
(1:500) and Alexa Fluor 588-conjugated donkey antirabbit IgG (1:500; Jackson
ImmunoResearch Lab, West Grove, Pennsylvania). After extensively washing, the slides were
mounted for fluorescence in Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole
(DAPI) (Vector Lab, Burlingame, California). Images were acquired using a Leica TCS SP2
A0BS Confocal System and processed on Leica Confocal Software version 2.6.1 (Leica,
Wetzlar, Germany). Detailed information of antibodies used in this study is listed in
Table 1 .
pacAd5.huCFTR-ECL4HA plasmid for 12 hours and cultured in the presence or absence of
nicotine for additional 24 hours. The cells were then fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) at room temperature for 15 minutes, washed in PBS for 3× 5
minutes, and permeabilized with 0.3% Triton X-100 for 10 minutes at room temperature.
Nonspecific antibody binding was blocked using 5% normal donkey serum in PBS for 1 hour at
room temperature, after which primary antibodies of mouse anti-CFTR and rabbit anti-HA
were applied at a 1:100 dilution in PBS and applied to probe proteins of interest by
incubating slides at 4°C overnight. The primary antibody binding was detected using the
Alexa Fluor 488-labeled donkey-anti-mouse immunoglobulin G (IgG) secondary antibody
(1:500) and Alexa Fluor 588-conjugated donkey antirabbit IgG (1:500; Jackson
ImmunoResearch Lab, West Grove, Pennsylvania). After extensively washing, the slides were
mounted for fluorescence in Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole
(DAPI) (Vector Lab, Burlingame, California). Images were acquired using a Leica TCS SP2
A0BS Confocal System and processed on Leica Confocal Software version 2.6.1 (Leica,
Wetzlar, Germany). Detailed information of antibodies used in this study is listed in
4
Immunofluorescent Staining of AECII and AECI Markers
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Immunofluorescent staining was applied to determine the expression of AECII cell marker surfactant protein C (SPC) and AECI cell marker aquaporin-5 (AQP-5). The membranes of 2-week ALI cultures were fixed in filtered 4% paraformaldehyde at room temperature for 15 min prior to be washed for 3×3 min with PBS. The cells were then permeabilized with 0.2% Triton X-100 for 20 min at room temperature, followed by blocking with 5% normal donkey serum in PBS at room temperature for 60 min, after which they were incubated with primary antibodies against SPC (1:1000, Merck Millipore, USA) or AQP-5 (1:500, Abcam, USA) in PBS at 4°C overnight. Following extensive washing for 3×10 min with PBS to remove primary antibodies, the membranes were incubated with Alexa Fluor 488-labelled donkey-anti-rabbit IgG secondary antibody (1:500, Jackson ImmunoResearch Laboratories, USA) at room temperature for 60 min. The stained membranes were then mounted on slides with Vectashield Mounting Medium containing DAPI (Vector Laboratories, USA), and covered with a coverslip after washing in PBS for 3×5 min. Images were acquired by Leica TCS SP2 A0BS Confocal System and processed on Leica Confocal Software v.2.6.1 (Leica, Germany).
5
Intracellular Signaling Dynamics in BCG-Infected THP-1 Cells
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THP-1 cells (1 × 105 cells/well) were seeded in 12-well plates with cell coverslips, transfected with siRNA-DUSP1, and cultured for 24 h post-transfection. Cells were infected with BCG (MOI = 10) and incubated for another 6 h. The cells were then washed 3 times with PBS and fixed with 4% paraformaldehyde for 20 min. The cells were washed and permeabilized with 0.5% TritonX-100 for 20 min, then blocked with 3% BSA for 1 h at room temperature. Antibodies were diluted in 3% BSA at dilution factors of DUSP1 (1:200), Cleaved-Caspase3 (1:200), Cleaved-PARP1 (1:200), p-p38 (1:100), p-JNK (1:100), p-ERK (1:200), and p-NF-κB p65 (1:200). 500 μL of the primary antibody was added to each well and incubated overnight at 4 °C. After washing three times with PBS, cells were incubated with fluorescein-coupled secondary antibody (1:1000) in PBS for 1 h at 37 °C, protected from light. Finally, cells were stained with a fluorescent blocker containing DAPI (ORIGENE, China) blocked, and images were acquired with a Leica TCS SP2 A0BS confocal system and processed on Leica confocal software (Leica, Germany).
6
Visualizing rCPB2 Toxin Binding
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The NCM460 cells cultured in collagen-coated cover slides were treated with rCPB2 toxin for different times (5 min, 30 min, 2 h, 4 h, 6 h, and 12 h) for evaluating the binding of rCPB2 to cell membranes. The rCPB2-treated cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min, washed in PBS for 3 × 5 min, and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. Nonspecific antibody binding was blocked using 5% normal donkey serum in PBS for 1 h at room temperature, after which primary antibodies against CPB2 toxin were applied at a 1 : 100 dilution in PBS and incubated at 4°C overnight. Primary antibody binding was detected using the FITC-labeled donkey-anti-mouse IgG secondary antibody (1 : 500) (Thermo, Rockford, USA). After extensive washing, cell membranes were mounted by Annexin A2 Polyclonal Antibody (1 : 200) (SanYing Biotechnology, China) as primary antibody, and Rhodamine- (TRITC-) conjugated goat anti-rabbit IgG as secondary antibody (1 : 250) (SanYing Biotechnology, China). After extensively washing, the slides were mounted for fluorescence in Vectashield Mounting Medium with DAPI (Thermo, Rockford, USA). Images were acquired using a Leica TCS SP2 A0BS Confocal System and processed on Leica Confocal Software v.2.6.1 (Leica, Germany).
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