RAW264.7 cells were plated on imaging slides (µ-Slide 12-well, glass bottom, Ibidi GmbH, Munich, Germany), followed by infection with KST0669 at an MOI of 10. Unbound bacteria were washed out with PBS followed by fixation with 2% paraformaldehyde at 4 °C for 20 min and permeabilization with 0.1% Triton-X100 for 20 min. The cells were then washed three times with PBS and blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich) in PBS for up to 2 h at room temperature. The cells were then incubated with rabbit anti-FMDV IgG, followed by staining with FITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich). The nuclei were stained with 150 ng/mL 4′,6-diamino-2-phenylindole (DAPI; Thermo Scientific). The slides were washed with PBS and mounted with mounting medium (Dako, Carpinteria, CA, USA). All images were captured using an Olympus CX41 fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Cx41 fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The CX41 fluorescence microscope is a laboratory equipment designed for the examination of fluorescently labeled specimens. It features a fluorescence illumination system and optical components that enable the visualization and analysis of samples under fluorescent conditions.
Automatically generated - may contain errors
Lab products found in correlation
Antifade reagent, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Fitc conjugated goat anti rabbit igg, by Merck Group (1 mentions)
4 6 diamino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
4 6 diamino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Ascent 2, by Thermo Fisher Scientific (1 mentions)
Axiovert 25, by Zeiss (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti serpine1, by Abcam (1 mentions)
Alexa fluor 488 conjugated anti igg, by Abcam (1 mentions)
Anti enos, by BD (1 mentions)
Anti casp9 antibody, by Proteintech (1 mentions)
Anti wnt2b antibody, by Bioss Antibodies (1 mentions)
Anti wnt7a antibody, by Bioss Antibodies (1 mentions)
Anti cdkn2a antibody, by Proteintech (1 mentions)
11 protocols using cx41 fluorescence microscope
1
Immunofluorescence Assay for FMDV Detection
2
Visualizing Intracellular Salmonella in RAW264.7 Cells
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Infected Salmonella cells were visualized by plating RAW264.7 cells on 15 μm Chamber 12-well glass slides (Ibidi GmbH), followed by infection with WT and KST0555 strains at a MOI of 10 at 37°C for 1 h. DMEM containing gentamicin (100 μg/ml) was used to eliminate extracellular bacteria. Following 2 or 18 h of infection, the cells were fixed with 4% paraformaldehyde for 30 min at 4°C and permeabilized with 0.1% Triton-X100 in PBS for 20 min. The cells were then washed three times with PBS and blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) in PBS for up to 2 h at room temperature. Intracellular Salmonella were detected by incubating the cells with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit Salmonella-specific antibodies (cat. no. ab20320; 1:1,000; Abcam) at room temperature for 30 min. The nuclei were stained with 150 ng/ml 4′,6-diamino-2-phenylindole for 5 min at 37°C (Thermo Fisher Scientific, Inc.). All images were captured using an Olympus CX41 fluorescence microscope (Olympus Corporation).
3
Multimodal Analysis of Neuronal Differentiation
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Morphological analysis and Perls’ Prussian blue staining were performed using an inverted microscope (Carl Zeiss, Axiovert 25, Milan, Italy) equipped with a 32X objective combined with a digital camera (Canon Powershot G8, Carl Zeiss, Milan, Italy). CX41 Olympus fluorescence microscope (Olympus Italia S.R.L., Segrate (MI), Italy) equipped with oil immersion objective (100X) lens, combined with a EPI LED Cassette (FRAEN, Settimo Milanese (MI), Italy) for excitation light and with digital camera (Infinity2, FRAEN, Settimo Milanese (MI), Italy), was employed for the fluorescence analysis. Measurement conditions were the following: 470 nm excitation (T% = 40), 505 nm dichroic beamsplitter, and 510 nm long pass filter. Flow cytometry analysis was applied to quantified the neuronal markers expression during the differentiation process using a two-laser flow cytometer (FACSCantoII, BD Biosciences, San Jose, CA, USA) combined with Diva Software. A microcentrifuge PICO 21 Thermo Fisher (Bioidea 2 Sas, Settimo Milanese (MI), Italy) was used to pellet the cells. ATP content, cell death and caspase 3/7 activity were quantified by a microplate fluorometer (Fluoroskan, Thermo Scientific, Milan, Italy) combined with PC software (Ascent 2.6, Thermo Scientific, Milan, Italy).
4
Fixation and Fluorescent Staining of hNLCs
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The hNLCs cell cultures were fixed in PF 4% (20 min at r.t.) and after two washing (PBS) were supravitally stained with the fluorescent nuclear dye, Hoechst 33342 (5 µM for 10 min at r.t.). Afterwards the hNLCs were washed (PBS before then H2O), let dry and scored under fluorescence microscope (CX41 Olympus fluorescence microscope equipped with a 40× objective). The microscopic fields were photographed and stored on PC.
5
Comet Assay for DNA Damage
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The intestinal mucosa was removed immediately after sacrifice of animals and transferred into the Roswell Park Memorial Institute (RPMI) medium containing 1 mM EDTA. The solutions were sieved by muslin cloth into Petri dishes to collect the cell suspension. Single cell gel electrophoresis (Comet assay) was performed in alkaline conditions as described by Singh et al. [39 ]. The DNA was stained by ethidium bromide and visualized under a CX41 fluorescence microscope (Olympus, Japan). The comets were scored at a magnification of 100X and images of 50 cells (25 from each replicate slide) for each sample were scored. Comet tail length (migration of DNA from the nucleus in μm) was chosen as the parameter to assess nuclear DNA damage and was automatically generated by the Komet 5.5 (USA) image analysis system.
6
Quantification of SERPINE1 in HPCs
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HPCs were incubated in media containing different concentrations of PAI-039 in DMSO for 48 h, fixed with 100% methanol for 5 min at −20°C, and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% bovine serum albumin, and 0.1% Tween for 2 h at room temperature. The HPCs were then incubated with rabbit anti-SERPINE1 (diluted 1 : 100, Abcam, Cambridge, UK) at 4°C overnight. After washing three times in PBS, the primary antibodies were reacted with the corresponding Alexa Fluor 488-conjugated anti-IgG (diluted 1 : 500, Abcam, Cambridge, UK) at 37°C for 30 min. Sections were examined under an Olympus CX41 fluorescence microscope (Olympus, Japan).
7
Arterial Extracellular Matrix Characterization
8
Comet Assay for Lymphocyte DNA Damage
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The single cell gel electrophoresis or comet assay was performed to assess DNA strand breaks in lymphocytes. Fully frosted glass slides were coated with 1% agarose and lymphocyte samples. After lysis, DNA was allowed to unwind for 30 min at 4°C in alkaline electrophoretic solution and subsequently electrophoresed for 20 min. Finally, the slides were neutralized, stained with ethidium bromide and analyzed under a CX41 fluorescence microscope (Olympus, Japan) [42 ]. The comets were scored at a magnification of 100x and images of 50 cells (25 from each replicate slide) for each sample were scored. Comet tail-length and olive tail moment were chosen as the parameter to assess nuclear DNA damage and were automatically generated by Komet 5.5 (USA) image analysis system.
9
Immunofluorescence Assay for CASP9
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The tissues were sectioned and embedded in paraffin. Sections were incubated overnight at 4°C with anti-CASP9 antibody (1:100 dilution; proteintech, China). Slides were washed with phosphate-buffered saline (PBS) and incubated with a goat anti-rabbit IgG secondary antibody conjugated with fluorescein isothiocyanate (ZSDB-BIO, China) for 30 min with washed slides. After washing with PBS, they were incubated with an antifade reagent (Invitrogen, United States). Staining was visualized to determine protein expression levels using an Olympus CX41 fluorescence microscope (Olympus, Japan). The analysis results were performed using Image J software.
10
Immunohistochemical Analysis of WNT Proteins in NSCLC
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Immunohistochemistry was performed in 20 NSCLC tissues and adjacent normal tissues. Paraffin-embedded sections were stained to determine the expression level of proteins. Sections were incubated overnight at 4°C with anti-WNT2B antibody (1:100 dilution; Bioss, China), or anti-WNT7A antibody (1:100 dilution; Bioss, China). After washing with phosphate-buffered saline (PBS), the slides were incubated with a goat anti-rabbit IgG secondary antibody conjugated to fluorescein isothiocyanate (ZSDB-BIO, China) for 30 mins. They were washed with PBS and then incubated with an antifade reagent (Invitrogen, USA). Finally, staining was observed using an Olympus CX41 fluorescence microscope (Olympus, Japan). The results of the analyses were performed with Image J software.
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