Morphology and viability of S. coelicolor M145 and ΔglnA3 mutant cells grown in a rich complex YEME–TSB (1:1) medium was analyzed in presence of polyamines. Samples were taken from every culture after 72 and168 h of growth, and obtained perpetrates were observed under phase-contrast microscope under 400× enlargement. To detect live and dead cells, SYTO9 and PI (propidium iodide) stains of the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) were used. The SYTO 9 green fluorescent stain labels cells with intact membranes, as well as those with damaged ones. PI penetrates cells with damaged membranes, decreasing SYTO 9 stain fluorescence when both dyes are present. Thus, in the presence of both SYTO9 and PI, cells with intact cell membranes appear fluorescent green whereas cells with damaged membranes appear red. The staining solution was prepared by mixing 0.75 μl of component A and B in 500 μl of water. Stained cells were analyzed by the fluorescence microscopy using Olympus BX60 microscope with an Olympus UPlanFl 100 × oil objective and an Olympus BX-FLA reflected light fluorescence attachment. Images were taken with the F-view II camera (Olympus), using TxRed and eGFP filter sets for detection of the fluorescent markers. The ImageJ was used for image processing. Significant number of images (10) was analyzed in a minimum of three independent culture analyses.
Bx fla
Manufactured by Olympus
Sourced in Japan
The BX-FLA is a fluorescence illuminator designed for Olympus BX series microscopes. It provides a stable and uniform light source for fluorescence observation and imaging. The BX-FLA supports a range of fluorescence filter sets to enable a variety of fluorescence techniques.
Automatically generated - may contain errors
Lab products found in correlation
Uplanfl 100 oil objective, by Olympus (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
F view 2 camera, by Olympus (1 mentions)
Rhodamine conjugated phalloidin solution, by Thermo Fisher Scientific (1 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
Jenaval, by Zeiss (1 mentions)
Axio vision le 4, by Zeiss (1 mentions)
Ds qi1mc, by Nikon (1 mentions)
Flexiperm, by Greiner (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Immunostep (1 mentions)
Alexa fluor 555 donkey anti mouse igg, by Beyotime (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Beyotime (1 mentions)
13 protocols using bx fla
1
Microscopic Analysis of S. coelicolor Morphology
2
Visualization of Osteoclast Differentiation
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Verification of pre-osteoclasts was confirmed by staining for tartrate resistant acid phosphatase (TRAP) and actin ring formation in wild-type and itgb3 deficient osteoclasts was visualized by rhodamine-phalloidin. TRAP staining was performed using the napthol-based method45 (link). Briefly, cells were incubated with 0.05 mol/L Acetate buffer, pH 5.0, containing 0.27 mmol/L naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO), 1% vol/vol N,N-dimethylformamide, 1.6 mmol/L Fast Red LB salt and 50 mmol/L sodium tartrate at 37°C for 10 minutes.
Following TRAP staining, cells were permeabilized with 0.1% Triton X-100 in PBS for 3–5 mins. After washing with PBS, cells were incubated with rhodamine-conjugated phalloidin solution (Molecular Probes, Carlsbad, CA) diluted 1/200 in PBS containing 1% BSA for 1 hr. Actin rings were detected by fluorescence microscopy (Olympus BX-FLA, Osaka).
Following TRAP staining, cells were permeabilized with 0.1% Triton X-100 in PBS for 3–5 mins. After washing with PBS, cells were incubated with rhodamine-conjugated phalloidin solution (Molecular Probes, Carlsbad, CA) diluted 1/200 in PBS containing 1% BSA for 1 hr. Actin rings were detected by fluorescence microscopy (Olympus BX-FLA, Osaka).
3
Cytogenetic Analysis of Rhinoleucophenga Species
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In this study ten male specimens of R. taquarussuensis R. neglectus
4
Cardiomyocyte Morphology and Fibrosis
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Following an overdose of isoflurane, hearts were extracted via thoracotomy, sectioned such that a basal sample was fixed in 4% paraformaldehyde and an apical sample was frozen in liquid nitrogen (SED: n = 6; EX: n = 5; EX + DOX: n = 6). Fixed sections were embedded in paraffin and stained with hematoxylin and eosin (H&E) and Masson's trichrome. Photographs of five cross sections for each animal were generated with a mounted digital camera (DS-Qi1Mc; Nikon, Tokyo, Japan) and light microscope (BX-FLA; Olympus, Shinjuku, Japan). To determine cardiomyocyte cross-sectional areas (CSA), H&E slides were analyzed by outlining round to cuboidal-shaped nucleated myocytes. Percent fibrosis was calculated as amount of collagen per myocardial cross section.
5
Immunodetection of TRPV4 in Sperm
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Sperm recovered from the cauda epididymis was dropped in 200 μl amounts onto slide
glasses equipped with flexiPERM (Greiner Bio-one, Tokyo, Japan) and adhered by
centrifugation (1500 rpm, 10 min). The sperm specimens were washed with
phosphate-buffered saline (PBS) after fixation with 4% paraformaldehyde for 10 min at
4ºC and washed again with PBS after 30 min treatment by 0.5% TritonX-100. The
specimen were treated with blocking buffer (Roshe, Tokyo, Japan) for 30 min and
reacted with rabbit anti-mouse TRPV4 antibody (1:100) (Alomone Labs, Jerusalem,
Israel) overnight at room temperature. After being washed with PBS, the specimen were
reacted with Cy3-labeled goat anti-rabbit IgG antibody (1:50) (Chemicon, NY, USA) for
30 min at room temperature and washed with PBS. The negative control was reacted with
normal rabbit immunoglobulin overnight at room temperature, and then subjected to the
following processing. We examined the specimens with a microscope (BX40, Olympus,
Tokyo, Japan) equipped with fluorescence device (BX-FLA, Olympus).
glasses equipped with flexiPERM (Greiner Bio-one, Tokyo, Japan) and adhered by
centrifugation (1500 rpm, 10 min). The sperm specimens were washed with
phosphate-buffered saline (PBS) after fixation with 4% paraformaldehyde for 10 min at
4ºC and washed again with PBS after 30 min treatment by 0.5% TritonX-100. The
specimen were treated with blocking buffer (Roshe, Tokyo, Japan) for 30 min and
reacted with rabbit anti-mouse TRPV4 antibody (1:100) (Alomone Labs, Jerusalem,
Israel) overnight at room temperature. After being washed with PBS, the specimen were
reacted with Cy3-labeled goat anti-rabbit IgG antibody (1:50) (Chemicon, NY, USA) for
30 min at room temperature and washed with PBS. The negative control was reacted with
normal rabbit immunoglobulin overnight at room temperature, and then subjected to the
following processing. We examined the specimens with a microscope (BX40, Olympus,
Tokyo, Japan) equipped with fluorescence device (BX-FLA, Olympus).
6
Apoptotic Cell Death Assessed by EB/AO and Annexin V-FITC
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Apoptotic cell death was assessed by ethidium bromide (EB) and acridine orange (AO) assay and by Annexin V-FITC Apoptosis Detection kit. For the first method, 40 µl of a mixture of AO (10 µg/ml) and EB (5 µg/ml) was added to cells treated at their respective IC50 for 12, 24 and 48 h. Cells with no treatment were used as a control group. The cells were observed in a fluorescence microscope (Olympus BX-FLA) and representative fields were photographed by digital camera (Olympus BX40, Japan). The Annexin V-FITC Apoptosis Detection kit was used according to the manufacturer's instructions. Briefly, 1×106 cells/well were incubated with the vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC50. After 16 h of incubation, cells were harvested, washed with PBS or annexin buffer (1:10) and double-stained with Annexin V-FITC solution and PI/RNase solution. After incubation, 300 µl of annexin buffer was added and fluorescence was analyzed by flow cytometry. Analysis was performed by FACSCantoII™, Becton Dickinson Immunocytometry Systems. The data were analyzed by Infinicyt Software version 1.7.0 (Cytognos S.L., Salamanca, Spain).
7
Apoptosis and Mitochondrial Membrane Potential
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Apoptotic cells were evaluated by the observation of morphological alterations with acridine orange (AO) and ethidium bromide (EB) (31) (link) and by the phosphatidylserine exposure with the Annexin V-FITC Apoptosis Detection kit (Immunostep, Salamanca, Spain). K562 and Jurkat cells (1 x 10 6 cells/well) were incubated for 12 hours with S1 at IC5024h. For the EB/AO staining, cells were resuspended in a 1:1 EB (5 μg/ml) and AO (10 μg/ml) solution and morphological characteristics were observed in a fluorescence microscope (Olympus BX-FLA) using a 40x objective. For the Annexin assay, cells were incubated with 5 μL of Annexin V-FITC according to the manufacturer's instructions and then acquired by flow cytometry as previously described.
Evaluation of the mitochondrial membrane potential (ΔΨm) K562 and Jurkat cells (1 x 10 6 cells/well) were incubated for 12 hours with S1 at IC5024h. Cells were then incubated with a MitoView (Biotium®, Fremont, USA) solution (1:10,000) for 30 minutes before flow cytometry acquisition (as previously described). The control groups (untreated cells) were considered as 100% of cells with an intact ΔΨm.
Evaluation of the mitochondrial membrane potential (ΔΨm) K562 and Jurkat cells (1 x 10 6 cells/well) were incubated for 12 hours with S1 at IC5024h. Cells were then incubated with a MitoView (Biotium®, Fremont, USA) solution (1:10,000) for 30 minutes before flow cytometry acquisition (as previously described). The control groups (untreated cells) were considered as 100% of cells with an intact ΔΨm.
8
Quantifying Cardiac Hypertrophy in Mice
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Five cross-sectional heart tissue images were captured for each mouse using a digital camera attached to a light microscope (BX-FLA; Olympus). A histopathologist assessed the presence of hypertrophic cells, counting their number across five high power fields (HPF) at 400x magnification, corresponding to an approximate area of 1 mm2. This method, adapted from Cree et al. and based on the World Health Organization (WHO) classification of tumors, involved enumerating hypertrophic cells characterized by increased size, bizarre shapes, irregular and hyperchromatic nuclei, as seen in H&E-stained sections. The total cell count across these fields was considered the hypertrophic cell count for each specimen.
9
Monitoring Autophagy in Fibroblasts
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The FLSs were seeded into 12-well plates at a density of 1 × 105 cells/well overnight. The MRFP-GFP-LC3 adenoviral vector stock, which was purchased from HanBio Technology Co. Ltd. (Shanghai, China), was adjusted to 50 MOI in 500 µL of culture medium. Then, the fibroblasts were incubated with adenoviruses for 2 h at 37 °C, and the culture medium was replaced with fresh medium containing 100 µg/mL aluminum particles. The cells were further cultured for 3 d. Autophagy was observed under fluorescence microscopy (Olympus BX-FLA, Japan) at 12, 24, 48, and 72 h, and the autophagic flux was evaluated by counting the number of GFP and mRFP puncta.
10
Histological Analysis of Synovial Tissue
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The synovial tissues of the hip joint, which were obtained during rTHA and THA operations, were fixed with 4% paraformaldehyde for 24 h. After 4, 8, and 12 weeks, the femurs and hips from each group of Sprague-Dawley (SD) rats were fixed and decalcified for 8 weeks at 4 °C. The tissues were embedded in paraffin and were cut in the coronal or sagittal plane centered over the area of particle erosion. Some of the sections were stained with H&E or Masson’s trichrome, and other sections were immunostained with the respective antibodies of interest. Negative controls were obtained without a specific antibody. The specimens were then observed and imaged under a light microscope (Olympus BX-FLA, Japan).
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