Eight-day-old seedlings of the wild type, rres1–1, and rres1–2 were incubated in liquid MS with or without 150 mM NaCl for 12 h before the seedlings were stained. For 3′, 3′-diaminobenzidine (DAB) staining, the seedlings were immersed in 1.0 mg/mL DAB (Sigma-Aldrich) dissolved in 50 mM Tris-HCl (pH 5.0) for 10 h and then washed three times with water. The roots were then photographed using a Carl Zeiss HBO100 microscope. For chloromethyl derivative of 2′,7′-dichlorodihydrofluorescin diacetate (CM-H2DCFDA) staining, seedlings were incubated in a buffer containing 10 μM CM-H2DCFDA (Sigma-Aldrich) at 37 °C in darkness for 30 min and then washed with distilled H2O to remove excess CM-H2DCFDA. The roots were then photographed using a Carl Zeiss HBO100 microscope. ROS levels were quantified based on the intensity of fluorescence by using ImageJ software (NIH, http://rsb.info.nih.gov/ij/ ).
Hbo100 microscope
Manufactured by Zeiss
The HBO100 microscope is a high-intensity mercury vapor illumination system designed for use with various microscope models. It provides a stable and uniform light source for illumination during microscopy applications.
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5 protocols using hbo100 microscope
1
ROS Levels in Arabidopsis Seedlings
2
Quantifying GFP Fluorescence in Salinity Stress
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Transgenic plants carrying ProDR5:GFP were crossed with rres1–1 and rres1–2 mutants, and F2 seedlings were screened on MS media containing 50 mg/L hygromycin. Meanwhile, hygromycin-resistant seedlings were genotyped for the rres1 mutations using specific primers (Table S1 ). The hygromycin-resistant and homozygous F3 seeds were sown on MS for 5 days and then were transferred to MS media supplemented with or without 100 mM NaCl, and grown for an additional 8 h. Seedlings were photographed by a Carl Zeiss HBO100 microscope. The intensity of fluorescence was quantified using ImageJ software.
3
Intracellular HIF-1α Immunostaining in Cells
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The intracellular HIF-1α staining of cells grown on coverslips in 6 well plates was carried out using the mouse monoclonal antibody for the HIF-1α subunit (Abcam ab16066). Cells were fixed with 4 % formaldehyde in PBS pH 7.4 and incubated with a 1:20 dilution of the monoclonal antibody overnight at 4 °C. The next day, cells were washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. All images were captured on a Zeiss LSM5 using the Zeiss HBO-100 microscope illuminating system. Images were processed using the Zen AIM application imaging program and converted to JPGs using Axiovision 40 Ver. 4.6.3.0. At least three biological repetitions were carried out.
4
Spheroid Formation Assay for HGF Activity
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Cells were seeded into a 6-well low attachment plate (Corning Inc. Life Science, Lowell, MA) at 5000 cells/well with DMEM supplemented with 10% FBS. 50 ng/mL recombinant human HGF (R&D, Minneapolis, MN), combined with HS20 or human IgG (50 μg/mL), was added to medium. The spheroid was photographed with a ZEISS HBO100 microscope on day 20. Images were acquired at 10x magnification. The volume of each spheroid is calculated by the formula v = 4πr3/3 (r represents the radius of spheroid).
5
Genetic Screening for EPS Mutants
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Screening of the exconjugants was mainly performed by PCR and sequencing. In addition, the plate-based assays were also used to screen for mutants whose EPS-related genes or cluster were deleted. For this, the colonies were streaked on EPS-inducing plates to check for the loss of slimy phenotype which indicated the elimination of EPS formation. An EPSglc plate was used for initial screening of the pepC gene or pep cluster knockout. In addition, an EPSsuc plate was used to screen for the ∆sacB ∆pepC double knockout mutants. The colonies were incubated at 30 °C overnight. Subsequently, the colonies were checked via colony PCR to verify the accuracy of the plate-based assay. Colony PCR was performed using primers which bind outside of the homologous regions provided as repair template. The resulting fragment was purified from the gel and sent for sequencing to confirm the modifications. Finally, gDNA was isolated from some of the colonies and again checked with PCR and sequencing to reconfirm the colony PCR results. Analysis with fluorescence microscopy was performed by using Carl Zeiss HBO 100 microscope. Microscopic images were processed with AxioVision imaging software.
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