Recombinant clones were generated using the MARCM system (Lee and Luo, 1999 (link)) or the temperature-sensitive esg flip-out (esgF/O) system (Jiang et al., 2009 (link)). MARCM adults of the desired genotype were subjected to three, 30-minute 37°C heat-shocks separated by 1h at room temperature. Animals were aged for ten or thirty days at 25°C for MARCM and 29°C for flip-out flies. Clonal size in MARCM or esgF/O experiments was determined by counting the number of nuclei labeled by DAPI on an Olympus BX51 epi-fluorescent microscope. Clone size was not determined for 30-day old esgF/O animals as no distinct clones cloud be identified.
Bx51 epifluorescent microscope
Manufactured by Olympus
Sourced in United States, Japan
The BX51 is an epifluorescent microscope manufactured by Olympus. It is designed for fluorescence imaging and observation. The BX51 utilizes epi-illumination, allowing for efficient fluorescence excitation and detection.
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Lab products found in correlation
Dm5500b wide field microscope, by Leica (1 mentions)
Anti ki67 ab15580, by Abcam (1 mentions)
Cellsens standard 1, by Olympus (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Dp71 camera, by Olympus (1 mentions)
Anodisc filter, by Cytiva (1 mentions)
Sybr green 1 dye, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Dp70 digital camera, by Olympus (1 mentions)
4 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Fluoromount g, by Beckman Coulter (1 mentions)
Sm2010r, by Leica (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Blocking buffer, by Thermo Fisher Scientific (1 mentions)
Hm430 sliding microtome, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Vegfa, by Proteintech (1 mentions)
Mmp 9, by Abcam (1 mentions)
18 protocols using bx51 epifluorescent microscope
1
MARCM and esg flip-out clonal analysis
2
Immunohistochemical Analysis of ORAI1, STIM1, DcR2, and Vimentin
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Immunohistochemical staining was performed on paraffin human prostate tissue arrays (Alenabio Company, Shanxi, China) and on the tumor xenograft tissues of NOD/SCID mice. The expression levels of ORAI1 and STIM1 were detected separately in these tissues using polyclonal rabbit antibodies raised against STIM1 or ORAI1 (ProSci Inc.) at a 1:100 dilution. The expression levels of ORAI1 or STIM1 in the tissue microarray were scored according to the percentage of ORAI1 or STIM1-positive cells in each whole area of prostate tissue and their staining intensity. Specifically, percentages ≤10%, 11%–30%, 31%–49% and ≥50% were scored as 1, 2, 3 and 4, respectively; non-significant brown, slight brown, moderate brown and deep brown staining intensities were scored as 1, 2, 3 and 4, respectively. The two scores were then added, and a score of 1–3 was considered weak, a score of 4–6 was considered moderate, and a score of 7–8 was considered strong.
The expression levels of DcR2 and Vimentin in tumor xenografts were detected separately with polyclonal rabbit antibodies raised against DcR2 and Vimentin (Proteintech Group Inc.) at a 1:100 dilution. Images were recorded using an Olympus BX51 Epi-fluorescent microscope (Olympus Co.).
The expression levels of DcR2 and Vimentin in tumor xenografts were detected separately with polyclonal rabbit antibodies raised against DcR2 and Vimentin (Proteintech Group Inc.) at a 1:100 dilution. Images were recorded using an Olympus BX51 Epi-fluorescent microscope (Olympus Co.).
3
Widefield Microscopy for Fluorescent Imaging
4
Immunofluorescence Staining of Paraffin-Embedded Tissue Sections
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Tissue sections on the slides were deparaffinized in xylene, rehydrated in ethanol, and rinsed in double-distilled water. Antigen retrieval was at 92C for 20 minutes using LabVision™ HIER Buffer L (Thermo Fisher Scientific Inc), slides were rinsed in 1xPBS, and tissue sections on the slides were incubated in normal goat serum from Vector Laboratories for 2 hours at room temp. Primary antibody from Abcam® (Anti-Ki67 ab15580) or (anti-alpha smooth muscle actin ab7817 and vimentin ab8069) or Santa Cruz biotechnology®, Inc. (HMG-1 sc-26351) was then added overnight at 4°C. On the following day, slides were rinsed in 1xPBS and the secondary antibody (Alexa Fluor® 594-conjugated AffiniPure Goat Anti-Rabbit for Ki67) or (Goat anti-mouse IgG1, Alexa Fluor® 594 for vimentin with Alexa Fluor® 488-conjugated AffiniPure goat anti-rabbit for alpha actin) or (Alexa Fluor® 594-conjugated AffiniPure Donkey anti-goat for HMG-1) was incubated on the slides for 2 hours at room temp. Slides were then rinsed in 1xPBS and Vector laboratories DAPI mounting medium was added to each slide. Tissue sections were viewed with an Olympus BX-51 epi-fluorescent microscope and images were photographed with an Olympus DP71 camera. These images were captured using Olympus cellSens Standard 1.11 software (Waltham, MA).
5
Enumerating Viruses and Bacteria via Epifluorescence
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Virus like particles (VLPs) and bacteria (in this paper Bacteria and Archaea), were enumerated by SYBR staining and epifluorescence microscopy (Noble and Fuhrman, 1998 ; Patel et al., 2007 (link)). Briefly, duplicate samples were fixed with 2% formamide (final concentration), filtered over a 25 mm 0.02 μm Anodisc filter (Whatman), stained using SYBR Green 1 dye (Molecular Probes-Invitrogen, Carlsbad, CA, USA) and mounted on slides with a glycerol, PBS, and p-phenylenediamine antifade solution. Slides were stored at −20°C until they were visualized using an Olympus BX51 epifluorescent microscope (Olympus America, Center Valley, PA, USA) to assess viral and bacterial abundance. Approximately 20 VLPs and bacteria were counted in 10 different viewing fields, averaged to the counts per grid box in the viewing field, and then converted to count ml−1.
6
In Vivo Imaging of Endozoic Cells
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Endozoic cells were imaged in vivo using a BX51 epifluorescent microscope equipped with a DP70 digital camera (Olympus) using the “chlorophyll” filter set (excitation: 480 nm, emission: 660 nm).
Four colonies of Mc and two colonies of Mf were collected and maintained in tanks with unfiltered, flow through sea water, for four weeks during June 2012. Colonies were exposed to natural light conditions. All colonies were examined for the percentage of the total colony that was visibly covered with mucus. When visible, samples of mucus were collected with a sterile syringe, incubated with SYBR Green nucleic acid stain as per the manufacturer’s instructions (Bio-Rad), and examined using an Olympus BX5100 epi-fluorescent microscope.
Four colonies of Mc and two colonies of Mf were collected and maintained in tanks with unfiltered, flow through sea water, for four weeks during June 2012. Colonies were exposed to natural light conditions. All colonies were examined for the percentage of the total colony that was visibly covered with mucus. When visible, samples of mucus were collected with a sterile syringe, incubated with SYBR Green nucleic acid stain as per the manufacturer’s instructions (Bio-Rad), and examined using an Olympus BX5100 epi-fluorescent microscope.
7
Diabetic Cell Adhesion Assay
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Adhesion of diabetic PCs and fibroblasts was measured in AMD3100 (50 ng/mL) ± PDGF-BB (100 ng/mL). PCs and fibroblasts (1×105 cells/chamber) were added to 4 well chamber slides (Fisher Scientific; Pittsburgh, PA) coated with fibronectin (5 μg/cm2) (Sigma) and incubated at 37°C for 2 hours. Following incubation, non-adherent cells were removed before adherent cells were fixed with 1% paraformaldehyde. Adherent cells were stained with DAPI (4′,6-diamidino-2-phenylindole) (VectaShield; Vector Laboratories, Burlingame, CA) and viewed on an Olympus BX51 epifluorescent microscope. Adobe Photoshop CS3 (Adobe Systems; San Jose, CA) was used to quantify the number of cells/random high-powered field (hpf) under 100× magnification. A total of 6 hpf/group was analyzed for comparison between experimental groups.
8
Immunohistochemical Analysis of c-Fos in Mouse Brain
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Immunohistochemistry was performed following previously described procedures (Wang et al., 2014 (link)). Briefly, mice were deeply anaesthesized and transcardially perfused with PBS, followed by 4% PFA in PBS. Brains were extracted and further fixed in 4% PFA overnight at 4°C followed by cryoprotection in a 30% PBS-buffered sucrose solution for 36 h. Coronal sections of 50 μm were cut using a freezing microtome (Leica SM 2010R). Sections were first washed in PBS (3 min × 10 min) and then blocked in 5% normal goat serum in PBST for 60 min at room temperature, followed by incubation with primary anti-c-fos antibody (rabbit, Santa Cruz Biotechnology, 1:5000) overnight at 4°C. Sections were then washed with PBS (3 min × 10 min) and incubated with fluorescent secondary antibody at room temperature for 1 h. After washing with PBS (3 min × 10 min), sections were mounted onto slides with Fluoromount-G (Beckman Coulter). Images were taken using an Olympus BX51 epifluorescent microscope.
9
Acridine Orange Mucus Staining
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AO solution (pH 3.5) was prepared by dissolving
20 mg of AO powder in 190 mL Na-acetate buffer (100 mL of 1 M N-acetate trihydrate + 90 mL 1 M HCl).68 (link) The slides (Superfrost Plus Gold Adhesion) with fixed mucus
samples were immersed in the AO solution for 2 min, rinsed in tap
water and dH2O, and cover-slipped with the Fluoroshield
mounting medium. AO mucus staining was analyzed using the U-MNIB2
and U-MWIG2 filter sets on the Olympus BX51 epifluorescent microscope
(Olympus, Japan).
20 mg of AO powder in 190 mL Na-acetate buffer (100 mL of 1 M N-acetate trihydrate + 90 mL 1 M HCl).68 (link) The slides (Superfrost Plus Gold Adhesion) with fixed mucus
samples were immersed in the AO solution for 2 min, rinsed in tap
water and dH2O, and cover-slipped with the Fluoroshield
mounting medium. AO mucus staining was analyzed using the U-MNIB2
and U-MWIG2 filter sets on the Olympus BX51 epifluorescent microscope
(Olympus, Japan).
10
Quantifying Cellular Markers Around Implants
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