Digital images of Candida cells were captured using an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software (Olympus,Warsaw, Poland). ImageJ software (http://rsbweb.nih.gov/ij/ ) was used to analyze the cell size. Cell size was expressed as arbitrary units [a.u.].
Bx61 fluorescence microscope
Manufactured by Olympus
Sourced in Japan, Germany, United Kingdom, United States, Canada, Spain, Italy
The BX61 fluorescence microscope is a versatile research-grade instrument designed for advanced microscopy applications. It features a high-quality optical system, a motorized and programmable stage, and a range of fluorescence illumination options. The BX61 is capable of performing various fluorescence imaging techniques, including wide-field and confocal microscopy, to support researchers in a variety of fields.
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Lab products found in correlation
Dp72 ccd camera, by Olympus (9 mentions)
Cell f software, by Olympus (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Vectashield, by Vector Laboratories (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions)
Hematoxylin eosin staining kit, by Wuhan Servicebio Technology (3 mentions)
Metavue acquisition software, by Molecular Devices (3 mentions)
Live dead yeast viability kit, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
3ccd digital camera c7780, by Olympus (3 mentions)
Hybrite, by Abbott (3 mentions)
Cytovision 7.2 spot counting system, by Leica (3 mentions)
Rabbit anti doublecortin dcx, by Abcam (2 mentions)
Minispin centrifuge, by Eppendorf (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Cellsens software, by Olympus (2 mentions)
Lsc lite version 2, by Leica (2 mentions)
166 protocols using bx61 fluorescence microscope
1
Measuring Candida Cell Size
2
Kinetics of Growth and Viability Assay
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For the kinetics of growth assay (Lewinska et al. 2011 (link)), cells at the logarithmic phase of growth were washed, diluted, suspended in YPD medium, and cultured at 30 °C. Their growth was monitored turbidimetrically at 600 nm in a microplate reader every 2 h during a 10-h period. Cell viability was estimated with a LIVE/DEAD® Yeast Viability Kit (Thermo Fisher Scientific, Poland) using the standard protocol according to the manufacturer’s instructions as described elsewhere (Lewinska et al. 2014a (link)). Briefly, cells at the logarithmic phase of growth were washed and stained with a mixture of FUN® 1 and Calcofluor® White M2R and inspected under an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. Typically, a total of 200 cells were used for the analysis.
3
Immunofluorescence Staining Protocol
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Cells were collected and fixed in 4% paraformaldehyde for 20 min at room temperature, rinsed in PBS twice and then in 5% BSA and 0.01% Triton X-100/PBS to permeabilize them and block protein-binding sites. The relevant primary and isotype control (as negative control) antibodies were added and cells were incubated at 4°C, overnight. Alexa-488 conjugated secondary antibodies were added at a dilution 1:500 for 2 h. Hoechst 33242 was used to stain nuclei (blue on images) and coverslips mounted on slides using mounting medium (Southern Biotech, Birmingham, AL, USA) for fluorescence. An Olympus BX61 fluorescence microscope was used to record images.
4
Smad9 and P-smad9 Protein Localization in Granulosa Cells
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Localization and expression of Smad9 and P-smad9 proteins in the GCs was evaluated by immunofluorescence (IF). Cells growing on the glass slide were washed in PBS, fixed in Immunol Staining Fix Solution (Beyotime Biotechnology) for 10 min, permeabilize with 0.5% Triton for 10 min, after that, blocked for 1 h at room temperature with 5% BSA. Then the GCs were incubated with antibodies against Smad9 (1:1000), P-smad9 (1:800) at 4°C for overnight. Cells were rinsed three times in PBS after primary antibody incubation and then were sequentially incubated with Alexa Fluor 555 donkey anti-rabbit secondary antibody (Beyotime Biotechnology, 1:1000) (Nie et al. 2012 (link)) for 1 h at room temperature. Finally, cells washed in PBS to omitting the antibodies mixture. After that, we used anti-fade Mounting Medium with DAPI (Solarbio, Beijing, China) to mount slides. Fluorescence images were acquired with an Olympus BX61 fluorescence microscope. Fluorescence images were recorded using a 40× objective lens.
5
Immunostaining of Cellular Proteins
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Cells were fixed with paraformaldehyde, permeabilized with digitonin, and further processed for immunostaining of cellular proteins with specific primary antibodies and appropriate corresponding fluorescence-labeled secondary antibodies, as described [17 (link)]. Where indicated, lipid droplets and nuclei were stained with 1 μg/ml Bodipy 493/503 (Invitrogen) and 1 μg/ml Hoechst 33342 (Invitrogen), respectively. Fluorescent signals were visualized using a BX61 fluorescence microscope (Olympus) and z-plane images at 0.5-μm intervals encompassing the depth of the cell were captured. Flat-field-corrected image stacks were deconvolved using Huygens software (Scientific Volume Imaging). Colocalization analysis was performed using the Coloc 2 plugin of ImageJ.
6
Quantifying Sperm DNA Fragmentation
7
Immunohistochemical Localization of Cell Wall Epitopes
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Panicle samples were fixed in formaldehyde–acetic acid–ethanol (FAA, 50% ethanol, 5% acetic glacial and 3.7% formaldehyde) and then embedded in paraffin for sectioning. Briefly, sections on glass slides were blocked with 3% BSA in PBS (pH 7.2) for 30 min. Then sections were washed with PBS and incubated with monoclonal antibody JIM5, LM18 (Plant Probes, 1:10 dilution) (www.plantprobes.net ) for 2 h at 37 °C. After washing with PBS, secondary anti-rat antibody conjugated to fluorescein-isothiocyanate (anti-rat/FITC, IgM, Bioss, 1:100 dilution) was applied for 1h at 37 °C in the dark. Finally, sections were washed with PBS and mounted in PBS/glycerol-based anti-fade solution (5% n-propyl gallate in 90% glycerol/10% PBS) for observation using an Olympus BX61 fluorescence microscope (Olympus, Japan).
8
Characterization of Neural Progenitor and Neuronal Populations
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hNPCs were fixed, washed, and blocked as previously described13 (link), being followed by a 1 h incubation (at 37 °C) with primary antibodies: nestin (1:200, Millipore, Billerica, MA, USA) to detect neural progenitors, rabbit anti-doublecortin (DCX; 1:500, Abcam, Cambridge, MA, USA) to detect immature neurons, mouse anti-rat microtubule associated protein-2 (MAP-2; 1:500, Sigma) and neuronal nuclei (NeuN; 1:100, Millipore) to detect mature neurons. Secondary antibodies namely, Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG, Life Technologies) and Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG, Life Technologies) were used for 1 h at 37 °C and then 10 min with 4-6-diamidino-2-phenylindole-dihydrochloride (DAPI; Life Technologies). Samples were observed under an Olympus BX-61 Fluorescence Microscope (Olympus, Hamburg, Germany). For this purpose, three coverslips and ten representative fields per condition were chosen and analyzed. In order to normalize the data between the different sets, the results are presented in percentage of cells. This was calculated by counting the cells positive for NeuN/MAP-2/DCX markers, and dividing this value by the total number of cells/field (DAPI-positive cells; n = 3).
9
3D Matrigel Vascular Network Assay
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VM was evaluated using a three-dimensional (3D) culture model containing growth factor-reduced Matrigel (BD Biosciences). Briefly, a volume of 50 μL growth factor-reduced Matrigel was plated in 96-well plates, and allowed to polymerize at 37°C for 30 minutes. Next, 2 × 104 cells were trypsinized and resuspended with serum-free medium, and then seeded onto the Matrigel layer. After 9 h incubation at 37°C with 5% CO2, each well was captured directly by an Olympus BX61 fluorescence microscope under a phase-contrast microscope (×100). The images were quantified for the mean area value of randomly selected vascular network meshes by ImageJ software. Each experiment was performed in triplicate.
10
TUNEL Assay for Apoptosis Detection in Pemphigus Vulgaris
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In this cross-sectional study, we selected 15 paraffin-embedded tissues with a
confirmed diagnosis of PV by hematoxylin and eosin staining, and 15 controls [skin
without histopathological alteration (normal variants)]. Pemphigus vulgaris
(patients) and tissue controls were between the third and fourth decades of
life.
One-micron-thick sections were cut and processed by TUNEL assay (transferase-mediated
dUTP nick end-labeling).
Deparaffinized and rehydrated tissue sections were subjected to antigen unmasking
with 0.1 M sodium citrate, pH 6.2; then, 100 ul DNAse-free proteinase K was placed
on each slide for 30 minutes. Next, 50 ul of TACS nuclease reaction mix
(streptavidin-HRP solution) (Roche) and 500 ul diluent (streptavidin) were added by
capillary action and incubated for 30 minutes.
Subsequently, 450 ul of enzyme solution and 50 ul of fluorescein-labeled solution
were prepared; 50 ul of each reagent was then added to the 15 slides and incubated
for 60 minutes at 37°C in a moist, dark chamber. Counterstaining was performed with
Evans blue, and a coverslip was placed, with fluorescent mounting medium (Dako
Fluorescent Mounting Medium, Cat. S3023, DakoCytomation). All washes between each
step were performed with TBS.
Reactions were examined under an Olympus BX61 fluorescence microscope. Positivity was
categorized dichotomously, and the results were analyzed by X2 test.
confirmed diagnosis of PV by hematoxylin and eosin staining, and 15 controls [skin
without histopathological alteration (normal variants)]. Pemphigus vulgaris
(patients) and tissue controls were between the third and fourth decades of
life.
One-micron-thick sections were cut and processed by TUNEL assay (transferase-mediated
dUTP nick end-labeling).
Deparaffinized and rehydrated tissue sections were subjected to antigen unmasking
with 0.1 M sodium citrate, pH 6.2; then, 100 ul DNAse-free proteinase K was placed
on each slide for 30 minutes. Next, 50 ul of TACS nuclease reaction mix
(streptavidin-HRP solution) (Roche) and 500 ul diluent (streptavidin) were added by
capillary action and incubated for 30 minutes.
Subsequently, 450 ul of enzyme solution and 50 ul of fluorescein-labeled solution
were prepared; 50 ul of each reagent was then added to the 15 slides and incubated
for 60 minutes at 37°C in a moist, dark chamber. Counterstaining was performed with
Evans blue, and a coverslip was placed, with fluorescent mounting medium (Dako
Fluorescent Mounting Medium, Cat. S3023, DakoCytomation). All washes between each
step were performed with TBS.
Reactions were examined under an Olympus BX61 fluorescence microscope. Positivity was
categorized dichotomously, and the results were analyzed by X2 test.
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