BEAS-2B cells were seeded on coverslips and incubated overnight in DMEM plus 10% FBS at 37°C with 5% CO2 in a humidified cell incubator. Cells were fixed using 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Cells were incubated with anti-XB13 (mouse IgG, homemade) and anti–Tks5 SH3#1 (rabbit IgG; EMD Millipore), primary antibody and secondary antibody conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR), or Oregon green 488 (Molecular Probes). Actin was stained using CytoPainter phalloidin-iFluor 405 reagent (Abcam). Coverslips were mounted onto glass slides with Dako fluorescence mounting medium (Dako, Mississauga, Canada). Images were obtained using an Olympus FluoView Confocal FV1000-ASW and analyzed by Olympus FluoView FV10-ASW (Olympus, Tokyo, Japan) and ImageJ (National Institutes of Health, Bethesda, MD).
Fluoview fv10 asw
Manufactured by Olympus
Sourced in Japan
The Fluoview FV10-ASW is a confocal laser scanning microscope system designed for high-resolution fluorescence imaging. It provides advanced features for capturing and analyzing detailed images of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000 confocal microscope, by Olympus (3 mentions)
Immu mount, by Thermo Fisher Scientific (2 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Cytopainter phalloidin ifluor 405 reagent, by Abcam (1 mentions)
Oregon green 488, by Thermo Fisher Scientific (1 mentions)
Fluoview confocal fv1000 asw, by Olympus (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
α sma, by Abcam (1 mentions)
Hoechst 33258, by Polysciences (1 mentions)
Pan cytokeratin, by Santa Cruz Biotechnology (1 mentions)
Imagej, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fv10 asw 3, by Olympus (1 mentions)
7 protocols using fluoview fv10 asw
1
Immunofluorescence Staining of BEAS-2B Cells
2
Cellular Uptake of TiO2-Alexa-Tf Nano-photosensitizers
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After grown to confluence, SNU-387 cells were plated at a density of 8000 cells/well on black-walled clear bottom 96 well plates and incubated at 37 °C for 24 h. Cells were then treated with TiO2-Alexa-Tf nano-photosensitizers. Both brightfield and fluorescence confocal microscopy were performed at 4, 24, and 72 h post treatment using an Olympus FV1000 confocal microscope using the AlexaFluor 633 excitation/emission channel. Fluorescence and bright-field image overlay with false color was performed using Fluoview FV10-ASW software from Olympus.
3
Immunohistochemical Analysis of Tissue Samples
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The tissues were dissected in Dulbecco’s phosphate buffered saline (PBS), fixed overnight at +4°C in 4% paraformaldehyde (PFA), washed briefly, dehydrated and embedded in paraffin and sectioned (6 µm). The tissue sections were stained with haematoxylin and eosin or used for immunohistochemistry. Antigen retrieval was enhanced by boiling the slides in 0.02 m citric acid buffer, pH 6, for 20 min or by incubating them in 0.04% pepsin, pH 2, for 30 min. followed by blocking with the appropriate serum for 1 h.
The primary antibodies used were Pax2 (Covance, PRB-276P) (1:100), α-SMA (Abcam ab5694) (1:100) and pan-cytokeratin (1:100) (Santa Cruz, sc-15367). These were incubated overnight at +4°C, washed with PBS, followed by 1 h of staining with the respective Alexa Fluor 488/546 or HRP-conjugated secondary antibody at a dilution of 1:1000. Hoechst 33258 (Polyscience, Inc.) was used to stain the nucleus. The specimens were mounted with Immu-Mount (Fisher Scientific), inspected using a confocal microscope and photographed (Olympus Fluoview FV10-ASW).
The activity of the floxed RosaR26R-derived β-galactosidase was identified as published (36 (link)), counterstained with eosin for 2 min and mounted with Immu-Mount (Fisher Scientific).
The primary antibodies used were Pax2 (Covance, PRB-276P) (1:100), α-SMA (Abcam ab5694) (1:100) and pan-cytokeratin (1:100) (Santa Cruz, sc-15367). These were incubated overnight at +4°C, washed with PBS, followed by 1 h of staining with the respective Alexa Fluor 488/546 or HRP-conjugated secondary antibody at a dilution of 1:1000. Hoechst 33258 (Polyscience, Inc.) was used to stain the nucleus. The specimens were mounted with Immu-Mount (Fisher Scientific), inspected using a confocal microscope and photographed (Olympus Fluoview FV10-ASW).
The activity of the floxed RosaR26R-derived β-galactosidase was identified as published (36 (link)), counterstained with eosin for 2 min and mounted with Immu-Mount (Fisher Scientific).
4
Transient Agrobacterium-mediated Transformation of Tobacco
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Leaves of N. tabacum cv. SR1 were used for Agrobacterium-mediated transient transformation. A single Agrobacterium colony was incubated in five ml of LB medium containing appropriated antibiotics, 50 μM acetosyringone, and 10 mM MES (pH 5.6). The culture was incubated at 28°C for 16 h and 1.5 ml was centrifuged at 13,000 rpm for 1 min. One ml of 10 mM MgCl2 was used to resuspend the pellet and the OD600 of the culture was adjusted to 0.2 and 100 μM of acetosyringone. N. tabacum leaves were infiltrated using a syringe without a needle. Mesophyll protoplasts were prepared as described by Carneiro et al. (1993) (link). After 4 and 5 days of infiltration, the microscope analysis was conducted using an Olympus FV1000 confocal microscope. Excitation filters were, respectively, for GFP and chlorophyll autofluorescence: 488 and 635 nm; Emission filters were, respectively, for GFP and chlorophyll autofluorescence: 510–550 nm and 670–700 nm. Images were obtained with the following software: Olympus FluoView FV10-ASW.
5
Drosophila Neuroanatomy Imaging Protocol
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Flies were raised on standard cornmeal/agar/yeast medium at 25°C and 60% humidity on a 12:12 light/dark cycle. The following fly stocks were used:
• MultiColor FlpOut (MCFO -1 ) (Bloom. #64085) and R14C11-GAL4 (Bloom.
#49256) (Figure 1A)
• MB465c-split GAL4 (Bloom. #68371), UAS-Brp-shortmStraw, UAS-GFP [55, 58] (Figure 1D)
• R25C01-GAL4 (Bloom. #49115), MultiColor FlpOut (MCFO -1 ) (Bloom. #64085), ChAT-LexA (Bloom. #60319), vGlut-LexA (Bloom. #60314), UAS-RFP, LexAop-GFP (Bloom. #32229) (Figure 6D-G ImmunoResearch Laboratories; #001-000-162) in PBST, and incubated in primary antibodies according to Table 1. Secondary antibodies were then applied for 24 hours.
Brains were then washed, ran through an ascending glycerol series (40%, 60%, and 80%), and mounted in VectaShield (Vector Labs H-1000). Images were acquired using either a 40x or 60x oil immersion lens on an Olympus FV1000 confocal microscope.
Images were processed in Olympus Fluoview FV10-ASW and ImageJ.
• MultiColor FlpOut (MCFO -1 ) (Bloom. #64085) and R14C11-GAL4 (Bloom.
#49256) (Figure 1A)
• MB465c-split GAL4 (Bloom. #68371), UAS-Brp-shortmStraw, UAS-GFP [55, 58] (Figure 1D)
• R25C01-GAL4 (Bloom. #49115), MultiColor FlpOut (MCFO -1 ) (Bloom. #64085), ChAT-LexA (Bloom. #60319), vGlut-LexA (Bloom. #60314), UAS-RFP, LexAop-GFP (Bloom. #32229) (Figure 6D-G ImmunoResearch Laboratories; #001-000-162) in PBST, and incubated in primary antibodies according to Table 1. Secondary antibodies were then applied for 24 hours.
Brains were then washed, ran through an ascending glycerol series (40%, 60%, and 80%), and mounted in VectaShield (Vector Labs H-1000). Images were acquired using either a 40x or 60x oil immersion lens on an Olympus FV1000 confocal microscope.
Images were processed in Olympus Fluoview FV10-ASW and ImageJ.
6
Immunofluorescent Staining of Testicular Tissue
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Adult male testes were fixed in 2% paraformaldehyde (PFA)/PBS for 3 h at 4°C, then washed briefly in PBS, placed in 30% Sucrose/PBS, followed by incubation in a 1:1 mixture of 30% Sucrose/PBS: OCT. Testes were embedded in OCT, frozen on dry ice, and sectioned (10 μm). Control and KO sections were placed on the same slide and stored at −80°C. The primary and secondary antibodies and dilutions are available in Table S2 . Nuclei were stained with DAPI.
Immunofluorescent staining with paraffin‐embedded specimens was also done as described previously41 with some modifications. Briefly, testes were dissected in PBS, fixed in 4% PFA overnight at 4°C, washed briefly in PBS, dehydrated through an ethanol series, embedded in paraffin, and sectioned (5 μm). After paraffin removal and rehydration, antigen retrieval was done by boiling slides in 0.01 M citric acid buffer, pH 6, for 20 min followed by blocking in PBS containing 5% fetal bovine and 5% goat serum for 1 h. The slides were stained with primary and secondary antibodies listed in Table S2 and Hoechst 33342 (Invitrogen). The specimens were mounted with Immu‐Mount (Fisher Scientific) and imaged by confocal microscopy (Olympus Fluoview FV10‐ASW) at Biocenter Oulu Tissue Imaging Center.
Immunofluorescent staining with paraffin‐embedded specimens was also done as described previously
7
Immunofluorescence Assay for PD-L1 Expression
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The cells were seeded in cover slips for 48 h. The glasses were taken out, placed in 6-well plates, and washed 3 times with 1× PBS. The cells were then fixed with 4% paraformaldehyde for 15 min. After blocking with 1% FBS in PBS for 1 h at RT, the cells were incubated with the primary antibody PD-L1 (CST, Cat. #86744, RRID: AB_2800088) at 4 °C overnight. After washing with 1× PBS 3 times, the cells were further incubated with a fluorescent secondary antibody (488, CST, Cat. #4412, RRID:AB_1904025) for 1 h at RT. The cells were then incubated in 4',6-diamidino-2-phenylindole (DAPI) for nucleus staining after washing 3 times with 1× PBS. Finally, immunofluorescent images were captured and analyzed by confocal acquisition software FV10-ASW 3.0 (Olympus Fluoview FV10-ASW, RRID: SCR_014215; Olympus, Tokyo, Japan).
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