An immunofluorescence analysis of transfected cells was performed 24 h after transfection62 (link). After staining with Hoechst 33342 and MitoTracker Red, cells were washed with DPBS, fixed in 4% paraformaldehyde in DPBS for 15 min, and permeabilized with 0.1% Triton X-100 in DPBS for 10 min at room temperature, followed by washing with 0.1% gelatin in DPBS36 (link). Permeabilized cells were incubated with a specific antibody in DPBS at room temperature for 1 h. After washing with 0.1% gelatin in DPBS, cells were incubated with the anti-IgG-FITC or anti-IgG-ALEXA594 antibody at room temperature for 1 h. After washing with 0.1% gelatin in DPBS, cells were observed using a Leica AF7000 fluorescence microscope (Leica, Solmser, Germany)36 (link). A line profile analysis was performed using the Line Profile Tool of Leica LAS AF software (Leica, Solmser, Germany). In this analysis, white lines in merged panels were converted to line profiles using the Line Profile Tool.
Af7000 fluorescence microscope
Manufactured by Leica
Sourced in Germany
The Leica AF7000 is a fluorescence microscope designed for high-performance imaging and analysis. It features advanced optics and illumination systems to deliver clear and reliable fluorescence imaging results.
Automatically generated - may contain errors
Lab products found in correlation
Las af software, by Leica (2 mentions)
Ultraview vox spinning disk microscope, by PerkinElmer (1 mentions)
Ab13970, by Abcam (1 mentions)
Cy3 conjugated anti rabbit, by Jackson ImmunoResearch (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Alexa fluor 488 conjugated anti chicken, by Jackson ImmunoResearch (1 mentions)
Ab99302, by Abcam (1 mentions)
6 protocols using af7000 fluorescence microscope
1
Immunofluorescence Analysis of Transfected Cells
2
Immunofluorescence Analysis of SAMM50 Localization
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Immunofluorescence analysis of transfected cells was performed 24 h after transfection [28 (link)]. After staining with Hoechst 33342 and MitoTracker Red, the cells were washed with DPBS, fixed in 4% paraformaldehyde in DPBS for 15 min, and permeabilized with 0.1% Triton X-100 in DPBS for 10 min at room temperature, followed by washing with 0.1% gelatin in DPBS. The permeabilized cells were incubated with anti-SAMM50 antibody (HPA034537) in DPBS for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were incubated with anti-Rabbit IgG-FITC antibody for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were observed using a Leica AF7000 fluorescence microscope (Leica, Solmser, Germany). Quantitative analysis of the mitochondrial localization of SAMM50 was performed by fluorescence microscopic observation of 50 immunofluorescence-positive (transfected) cells. The extent of mitochondrial localization was expressed as a percentage of the number of cells in which selective localization to mitochondria, localization to both mitochondria and cytoplasm, and selective localization to the cytoplasm was observed against the total number of transfected cells. Data are expressed as mean ± SD for 5 independent experiments.
3
Immunofluorescence Analysis of Transfected Cells
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Immunofluorescence analysis of transfected cells was performed 24 h after transfection [37 (link)]. After staining with Hoechst 33342 and MitoTracker Red, the cells were washed with DPBS, fixed in 4% paraformaldehyde in DPBS for 15 min, and permeabilized with 0.1% Triton X-100 in DPBS for 10 min at room temperature, followed by washing with 0.1% gelatin in DPBS. The permeabilized cells were incubated with a specific antibody in DPBS for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were incubated with anti-rabbit IgG-FITC antibody for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were observed using a Leica AF7000 fluorescence microscope (Leica, Solmser, Germany).
4
Immunofluorescence Analysis of Transfected Cells
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Immunofluorescence analysis of transfected cells was performed 24 h after transfection52 (link). After staining with Hoechst 33342 and MitoTracker Red, Nile Red, or Lipi-Green, the cells were washed with DPBS, fixed in 4% paraformaldehyde in DPBS for 15 min, and permeabilized with 0.1% Triton X-100 in DPBS for 10 min at room temperature, followed by washing with 0.1% gelatin in DPBS. The permeabilized cells were incubated with a specific antibody in DPBS for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were incubated with anti-IgG-FITC or anti-IgG-ALEXA594 antibody for 1 h at room temperature. The concentration of fluorescent probes and 1st- and 2nd-antibodies used in the immunofluorescence analysis was summarized in supplementary Table S6 . After washing with 0.1% gelatin in DPBS, the cells were observed using a Leica AF7000 fluorescence microscope (Leica, Solmser, Germany). Line profile analysis was performed using the Line Profile Tool of Leica LAS AF software (Leica, Solmser, Germany). For this analysis, the white lines in merge panels were converted to line profiles using the Line Profile Tool.
5
Dynamics of Sec Body Formation and Disassembly
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Time lapse of Sec body formation and disassembly was performed on S2 cells stably expressing GFP-Sec23. For Sec body formation, cells were incubated in KRB (t = 0) at 26°C. For Sec body disassembly, cells were starved for 4 hr and further incubated in Schneider's (t = 0) at 26°C. Cells were viewed with a Leica AF7000 Fluorescence microscope. 10 z planes with a z step of 0.7 μm of were recorded every 3 min.
The FRAP experiments were performed on cells expressing GFP-Sec23, ΔNC1-Sec16-sfGFP, or FMR1-sfGFP for 1.5 hr (expression induced with CuSO4) followed by incubation in Schneider's for 1 hr and starvation in KRB for 4 hr. Sec bodies and Stress Granules were entirely or partially (half) photobleached using a 488 nm laser at 100% laser power for 750 msec. FRAP was recorded using a PerkinElmer Ultraview VoX spinning disk microscope with the volocity software. Fluorescence recovery was recorded every 10 ms for the first 14 s after bleaching, and thereafter every 10 s for 2 min.
The FRAP experiments were performed on cells expressing GFP-Sec23, ΔNC1-Sec16-sfGFP, or FMR1-sfGFP for 1.5 hr (expression induced with CuSO4) followed by incubation in Schneider's for 1 hr and starvation in KRB for 4 hr. Sec bodies and Stress Granules were entirely or partially (half) photobleached using a 488 nm laser at 100% laser power for 750 msec. FRAP was recorded using a PerkinElmer Ultraview VoX spinning disk microscope with the volocity software. Fluorescence recovery was recorded every 10 ms for the first 14 s after bleaching, and thereafter every 10 s for 2 min.
6
Immunostaining of EGFP and Tbx3-EGFP Embryos
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EGFP transgenic E17.5 embryos and Tbx3-flag-EGFP knock-in E15.5 embryos were cryoprotected in 20% sucrose/PBS and frozen in optimal cutting temperature compound. The samples were sectioned and subjected to immunostaining. The tissue samples were fixed with 4% paraformaldehyde, followed by permeabilisation with 0.5% Triton X-100 in TBS for 15 min at room temperature. Next, the sections were blocked with 5% bovine serum albumin at room temperature for 1 h, incubated with primary antibodies at 4 °C overnight, washed and then incubated for 1 h with secondary antibodies [Alexa Fluor 488-conjugated anti-chicken or Cy3-conjugated anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA)]. The following primary antibodies were used: anti-GFP (chicken, 1:2000, ab13970; Abcam) and anti-Tbx3 (rabbit, 1:200, ab99302; Abcam). The images of EGFP transgenic and Tbx3-flag-EGFP knock-in embryos were acquired using a Leica AF7000 fluorescence microscope and a Leica SP8 confocal microscope, respectively.
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