Cells were fixed in 4% paraformaldehyde (PFA) in cytoskeletal stabilization buffer (10 mM PIPES, pH 6.8, 100 mM KCl, 300 mM sucrose, 2 mM EGTA, 2 mM MgCl2) on ice for 30 min and subsequently treated with 50 mM NH4Cl for 10 min. Permeabilization was then performed with 0.2% Saponin/0.1% bovine serum albumin in Tris-buffered saline. Confocal images were taken using a Zeiss LSM-510 META inverted microscope and Zeiss LSM-710 FCS inverted microscope driven by ZEN software (ZEN 2009; Zeiss).
Lsm 510 meta inverted microscope
Manufactured by Zeiss
Sourced in Germany
The LSM-510 META inverted microscope is a high-performance imaging system designed for advanced scientific research. It features a versatile inverted configuration, allowing for a wide range of sample handling and observation techniques. The microscope integrates the META (Multi-Photon Excitation and Tunable Emission) technology, providing enhanced spectral resolution and flexibility in fluorescence imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 fcs inverted microscope, by Zeiss (2 mentions)
Anti lc3b, by Merck Group (1 mentions)
Anti pecam 1, by Merck Group (1 mentions)
Ab6717, by Abcam (1 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Donkey anti mouse fitc, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Lipofectamine ltx plus, by Thermo Fisher Scientific (1 mentions)
8 protocols using lsm 510 meta inverted microscope
1
Fixation and Permeabilization of Cells
2
Immunofluorescent Staining of Human PAECs
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The slides of human PAECs were prepared and then fixed with 40 g/l polyformaldehyde at room temperature for 15 min, washed three times with PBS, and sealed with 10% goat serum. Next, the slides were incubated overnight at 4°C with anti-LC3B (L7543; 1:100; Sigma-Aldrich, Merck KGaA; anti-rabbit) and anti-PECAM-1 (cat. no. sc-18916; 1:100; Santa Cruz Biotechnology, Inc.; anti-rat). After being washed with PBS three times, the tissues were subjected to further incubation with fluorescein isothiocyanate-labeled secondary antibodies (cat. no. Ab6717; 1:500; Abcam; anti-rabbit) and Cy3-labeled IgG secondary antibodies (cat. no. Ab6939; 1:500; Abcam; anti-rat) and incubated with protection from light for 60 min at room temperature. Confocal imaging was performed using an LSM510 Meta inverted microscope with a confocal laser scanning head (Carl Zeiss AG). Digital images were collected for analysis (SPOT; Diagnostic Instruments, Inc.). ImageJ (version 1.46; National Institute of Health) was employed to analyze the fluorescence intensity and calculate the fluorescence intensity value.
3
Microscopy Protocols for RPTPσ Staining
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Cells were fixed with 10% trichloroacetic acid on ice for 15 min for RPTPσ staining or fixed in 4% paraformaldehyde in cytoskeletal stabilization buffer (10 mM PIPES pH 6.8, 100 mM KCl, 300 mM sucrose, 2 mM EGTA, 2 mM MgCl2) on ice for 30 min and subsequently treated with 50 mM NH4Cl for 10 min. Permeabilization was then performed with 0.2% saponin/0.1% bovine serum albumin in Tris-buffered saline. Confocal images were taken using a Zeiss LSM-510 META inverted microscope and Zeiss LSM-710 FCS inverted microscope driven by ZEN software (ZEN 2009; Zeiss) and an Elyra microscope system equipped with a C-Apo 63×/1.2-W Korr objective for structured illumination microscopy.
4
Visualization of GmCIF1 and GmC/VIF2 Localization
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The coding sequences (omitting the stop codon) of GmCIF1 and GmC/VIF2 were amplified by PCR, including the primers containing the Gateway (Invitrogen) attB1 and attB2 recombinant sites (see Supplementary Table S2). The PCR products were recovered by using the GeneJET PCR purification kit (Thermo Scientific) and then inserted into the pDONR201 donor plasmid, sequenced, and subsequently recombined with the binary destination vector pB7YWG2.0, yielding the pB7GmCIF1-YFP and pB7GmC/VIF2-YFP constructs. Tobacco leaves were co-infiltrated with A. tumefaciens (C58C1) harboring pB7GmCIF1-YFP (or pB7GmC/VIF2-YFP) and a cell wall-localization marker pK7BvCWI-1RFP (Rosenkranz et al., 2001 (link)). The infiltrated region of the leaves was analyzed by CLSM. Images were taken with a Zeiss LSM 510 Meta inverted microscope. The yellow fluorescent protein (YFP) was excited with a 514-nm laser line, and the emitted fluorescence was collected using a 530–600-nm band pass filter. The red fluorescent protein (RFP) was excited with 543-nm laser line and the emitted fluorescence was collected with a 560-nm long pass filter.
5
Cloning and Visualization of GmCWI4
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Coding sequences (stop codon removed) of GmCWI4 were amplified by PCR using primers containing the Gateway attB1 and attB2 recombinant sites (Table S2 ). The amplified fragments were recovered by using the QIAquick PCR purification kit (Qiagen, Germany) and then inserted into pDONR201 donor vector. After sequencing, the donor vector subsequently recombined with the destination vector pK7RWG2.0, yielding the GmCWI4-RFP constructs. Onion epidermis was bombarded with A. tumefaciens (C58C1) harboring pK7RWG-GmCWI4 and a cell wall-localization marker pMDC32-SecGFP. Additional YFP fusion construct, pB7YWG-GmCWI4 was introduced into Arabidopsis plants that were transformed with membrane marker (pK7RWG-AtRLP44). Transfected regions of onion epidermis and root tip of four days transgenic Arabidopsis were analyzed by a LSM 510 Meta inverted microscope (Zeiss, Jena, Germany). The yellow fluorescent protein (YFP) was excited with a 514-nm laser line, and the emitted fluorescence was captured while using a 530-600 nm band pass filter. The red fluorescent protein (RFP) was excited with 543-nm laser line and the emitted fluorescence was collected with a 560-nm long pass filter.
6
Quantifying Telomere Dysfunction via γH2AX
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Fibroblasts were grown on sterile slides for several days. Cells were then fixed with 4% PFA, permeabilized with 0.5% Triton in PBS, and blocked for 1 h at 37°C in 5%FBS/1xPBS. Following these treatments, slides were incubated overnight at 4°C with the mouse anti phosphorylated γH2AX antibody (#05-636,; EMD Millipore) diluted 1:300 in 3%FCS/0.1% triton/1xPBS. Slides were then washed three times for 5 min in 0.1% Triton/PBS (PBST) at room temperature, and incubated with a secondary antibody, donkey anti-mouse-FITC (715–095-150, Jackson ImmunoResearch Laboratories, INC.) diluted 1:300 in 3% FCS/0.1% Triton X-100/1xPBS for 1 h at 37°C. After three washes in PBST, a second 10-min fixation with PFA was performed, followed by three 5-min washes with PBS and three dehydration steps, each for 5 min in EtOH (70, 90, and 100%), all at room temperature. The telomere-FISH protocol was then continued as previously described (Yehezkel et al., 2008 (link)) using a PNA-(CCCTAA)3 oligo conjugated to Cy3. For TIF analysis images were captured on a confocal LSM 510 Meta Inverted Microscope (Zeiss). At least 20 nuclei were analyzed for each cell type by IMARIS software (BITPLANE) on z stack projection images. The γH2AX foci were called as surfaces, and a co-localization channel was built on telomere and foci signals after which the percent of telomeres positive for co-localization was determined.
7
Immunocytochemistry of Neuronal Cell Lines
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HEK293, neuro2a cells and dissociated DRG neurons were cultured on glass coverslips previously treated with HBSS + 25% poly-L-ornithine and laminin (Sigma, Oakville, ON, Canada) and placed on either 35 mm dishes or a 24-well plate. Before immunostaining, cells were washed twice and fixed with 4% paraformaldehyde (PFA) for 15 min at 37 °C. Next, cells were permeabilized by adding 5% bovine serum albumin (BSA) (Sigma, Oakville, ON, Canada) with 0.3% Triton X-100 (Sigma, Oakville, ON, Canada) for 1 h at room temperature. Cells were incubated overnight with primary antibody in 1% BSA with 0.01% Triton X-100. On the next day, cells were washed three times, for 20 min each, with PBS and then incubated with the secondary antibody for 1 h at room temperature. Cells were washed for 30 min in PBS and imaged using a Zeiss LSM-510 Meta inverted microscope.
8
FRET Monitoring of PKCε Dynamics
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The vector for FRET probe with CFP and YFP at the N-and C-terminal ends of PKCε, respectively, was constructed and transfected into PC-12 cells using a Lipofectamine LTX-PLUS (Invitrogen, Carlsbad, CA, USA). Forty-eight hours later FRET monitoring was carried out with Zeiss LSM510 META inverted microscope (Oberkochen, Germany) by the method as described previously [14] . The CFP and YFT fluorescent signals were detected at an absorbance of 474 and 506 nm, respectively, using an excitation light of 458 nm. The FRET ratio (YFP signal intensity/CFP signal intensity) was calculated using an ImageJ software (National Institutes of Health, USA).
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