Fluorescent in situ hybridization (FISH) was performed on formalin-fixed, paraffin-embedded tumor tissue to detect deletion of chromosome 1p and 19q [15 (link)]. 4-μm-thick sections were deparaffinized, treated with sodium thiocyanate and followed by digestion with pepsin solution at 37°C. Dual-color-probe hybridization was then performed using Vysis 1p36/1q25 and 19q13/19p13 FISH Probe Kit (Abbott Molecular) and the spectrum-green-probe was labeled on chromosome 1q and 19p, respectively. Both probes and target tumor DNA were denatured in an 80°C oven for 30 minutes and followed by an overnight incubation at 37°C. Nuclei were counterstained with Vectashield mounting medium containing 4′, 6-diamidino-2- phenylindole (Vector Laboratories, Burlingame, CA, USA) and the number of FISH signals was assessed under a Zeiss Axioplan fluorescence microscope (Carl Zeiss Microscopy LLC, NY, USA) equipped with a triple-pass filter (DAPI/Green/Orange). Hybridizing signals of at least 100 non-overlapping nuclei were enumerated and a sample will be considered as 1p or 19q deleted when more than 25% of counted nuclei presented one target (orange) signal and two reference (green) signals [2 (link)].
Axioplan fluorescence microscope
Manufactured by Zeiss
Sourced in Germany, United States, United Kingdom
The Axioplan fluorescence microscope is an advanced optical instrument designed for high-performance fluorescence imaging. It features a modular design and a range of specialized illumination and detection systems to enable detailed analysis of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Poly l lysine (pll), by Merck Group (4 mentions)
Cy3 conjugated igg antibodies, by Jackson ImmunoResearch (4 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 goat or donkey igg antibodies, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
A11029, by Thermo Fisher Scientific (2 mentions)
Axiocam 503 mono microscope camera, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fitc labeled donkey anti goat igg, by Proteintech (2 mentions)
Ikarus digital imaging systems, by MetaSystems (2 mentions)
S3023, by Zeiss (2 mentions)
Fluorescence mounting media, by Agilent Technologies (2 mentions)
Plin1 antibody, by Cell Signaling Technology (2 mentions)
Vysis 1p36 1q25 and 19q13 19p13 fish probe kit, by Abbott (1 mentions)
Fitc labeled goat anti rabbit igg antibody, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Mab2018, by R&D Systems (1 mentions)
Anti nanog af1997, by R&D Systems (1 mentions)
77 protocols using axioplan fluorescence microscope
1
FISH Analysis for 1p/19q Deletion
2
Expressing Recombinant pVAX-TgCDPK2 in HEK 293-T Cells
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The recombinant pVAX-TgCDPK2 plasmids were transfected into HEK 293-T cells using the LipofectamineTM 2000 reagent (Invitrogen, USA), as indicated by the manufacturer. Forty-eight hours after transfection, the expression of pVAX-TgCDPK2 was examined by indirect immunofluorescence (IFA). Briefly, HEK 293-T cells were fixed with 4% paraformaldehyde and then incubated with goat anti-T. gondii tachyzoites polyclonal antibody (1:50). Then, fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat IgG antibody (1:1000; Sigma) was added. The specific fluorescence was imaged through a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany). As a negative control, the HEK 293-T cells were transfected with pVAX I.
3
Pluripotency Marker Characterization of Stem Cells
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To demonstrate pluripotency markers in the undifferentiated cells, live cells were fixed and permeablized before incubated with primary antibodies, anti-Nanog (AF1997, R&D Systems), SOX2 (MAB2018, R&D), Oct4 (MAB4401, Millipore) and Tra-1-81 (560161, BD Biosciences) antibodies in 5% BSA overnight, followed by secondary antibodies conjugated with Alexa-Fluor-488 (A-11008, or A-11029, ThermoFisher Scientific). Slides were covered by anti-fade mounting medium with DAPI (H-1200, Vector Laboratories) and visualized under Zeiss Axioplan fluorescence microscope (Carl Zeiss, NY).
To detect the surface markers by flow cytometry, cells were prepared and stained as previously described8 (link)9 (link) before reading with the fluorescent-activated cell-sorting facility (FACSCalibur, BD) of phycoerythrin (PE)-conjugated SSEA4 (560128, BD Biosciences) and Tra-160 (560884, BD Biosciences), or allophycocyanin (APC) or PE-conjugated CD31 (555446, BD Biosciences), CD34 (555824, BD Biosciences), CD44 (559942, BD Biosciences), CD73 (550257 BD Biosciences), CD105 (17-1057, eBioscience) and CD146 (550315, BD Biosciences). Staining of intracellular protein osteocalcin (IC1419P, R&D) was performed according to the manufacturer’s recommended protocol. These flow cytometry data was analyzed with FlowJo software (Tree Star, Ashland, OR).
To detect the surface markers by flow cytometry, cells were prepared and stained as previously described8 (link)9 (link) before reading with the fluorescent-activated cell-sorting facility (FACSCalibur, BD) of phycoerythrin (PE)-conjugated SSEA4 (560128, BD Biosciences) and Tra-160 (560884, BD Biosciences), or allophycocyanin (APC) or PE-conjugated CD31 (555446, BD Biosciences), CD34 (555824, BD Biosciences), CD44 (559942, BD Biosciences), CD73 (550257 BD Biosciences), CD105 (17-1057, eBioscience) and CD146 (550315, BD Biosciences). Staining of intracellular protein osteocalcin (IC1419P, R&D) was performed according to the manufacturer’s recommended protocol. These flow cytometry data was analyzed with FlowJo software (Tree Star, Ashland, OR).
4
Examining pVAX-PF Protein Expression
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HEK293 cells were transfected with the pVAX-PF or the empty pVAX I (negative control) using lipofectamine™ 2000 reagent (Invitrogen) following the manufacturer’s instructions. The expression of pVAX-PF plasmid was examined by the indirect immunofluorescence assay (IFA) at 48 h after transfection. Briefly, the HEK293 cells were fixed with acetone and then perforated with 0.1% Triton-X-100 in PBS. The cells were incubated with goat polyclonal antibodies against T. gondii tachyzoites (1: 50 in PBS) at 37 °C for 60 min, followed by fluorescein isothiocyanate (FITC)-labeled anti-goat IgG antibodies (Abcam, UK) diluted 1: 2000 in PBS. The specific fluorescence was imaged using a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany).
5
Evaluating Pro-Angiogenic Potential of Differentiated Cells
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In order to evaluate pro-angiogenic potential of these osteogenic differentiated cells from BM-MSCs, hESCs, UCBiPSCs and PBiPSCs, conditioned medium of these cells was collected and applied to stimulate tubulogenesis of human umbilical veil endothelial cells (HUVECs) in vitro. Briefly, the same amount of differentiated cells (p5 ~ 7) were re-plated and cultured overnight to ensure cell attachment to culture dish before switching the culture medium to serum free medium for an additional 48 hours; the conditioned media was collected and filtered to culture HUVECs on growth factor-reduced Matrigel (Corning). 4 hours later, the live HUVECs were stained with calcein AM (Life Technologies, NY) and tubulogenesis was visualized under Zeiss Axioplan fluorescence microscope (Carl Zeiss, NY). Total tube length and branching points were quantitatively analyzed by ImageJ software (NIH open resource).
6
Dystrophic and Bortezomib-Treated Muscle Analysis
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Quadriceps and triceps muscle from dy2J/dy2J, bortezomib-treated dy2J/dy2J, wild-type (WT) and bortezomib-treated WT mice were dissected after euthanasia and frozen in optimal cutting temperature compound (Tissue-Tek OCT; Sakura Finetek, Torrance, CA) in liquid nitrogen. Transverse cryosections of 7 μm were stained with hematoxylin and eosin (H&E), Masson’s trichrome (using an HT15 commercial kit; Sigma-Aldrich, St. Louis, MO) or biotinylated wheat germ agglutinin (WGA), which was detected with fluorescein avidin D (Vector Laboratories, Burlingame, CA). Sections were also processed for immunofluorescence analyses according to standard procedures with rat monoclonal anti-tenascin-C (MTn15) [11 (link)] and rat monoclonal anti-CD11b (M1/70, BD Pharmingen, San Diego, California). Anti-tenascin-C, anti-CD11b and WGA stained sections were analyzed and images captured with a Zeiss Axioplan fluorescence microscope (Carl Zeiss Microscopy, Jena, Germany) using an ORCA 1394 ER digital camera (Hamamatsu Photonics, Hamamatsu City, Japan) and Openlab software version 3 (Improvision, Coventry, UK).
7
Fluorescence Microscopy Protocol
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Samples were examined with a Zeiss Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with a differential interference contrast (DIC) optical system and an Axiocam MRc5 digital camera (Zeiss). For fluorescence images the same exposure time was used. Moreover, a Zeiss Stemi 2000-C stereomicroscope equipped with a Jenoptik ProgRes3 (Jenoptik, Jena, Germany) digital camera was also used.
8
Plasmid Transfection and Immunofluorescence Assay
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The recombinant plasmid pVAX-CDPK1 transfection into Marc-145 cells was performed using lipofectamine™ 2000 reagent (Invitrogen) as instructed by the manufacturer. In brief, forty eight hours post-transfection, cells were processed for indirect immunefluorescence assay (IFA) followed by incubation with goat anti-T. gondii tachyzoites polyclonal antiserum and a FITC-labeled donkey-anti-goat IgG antibody (Proteintech Group Inc., Chicago, USA). The specific fluorescence was examined through a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany). Marc-145 cells transfected with empty pVAX I served as the negative control.
9
Evaluating Protein Hemolytic Potential and AMP Toxicity
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The hemolytic potential of the protein and peptide variants was tested on Columbia blood agar plates (VWR). Sterile filter discs (Ø6 mm) were placed on the agar plates and loaded with 20 μg (∼10 μL) of each protein and peptide. Sterile water and 20% Triton-X 100 served as negative and positive control, respectively. The plates where incubated for 24 h at 37°C before evaluation.
AMP toxicity was determined on HKC and HDF. Keratinocytes and fibroblasts were isolated and grown in CnT-07 (CellnTEC) or R10 (Supplementary TableS2 ), respectively, as described previously (Blunder et al., 2017 (link)). Fluorescence viability staining was performed on HKC and HDF cells grown on chambered cell culture slides (Falcon). 4 × 103 cells per well were seeded and grown at 37°C and 5% CO2 before AMPs were added at 30 μM for 24 h. Cells were washed with PBS and the fluorescent dyes PI (1 μg mL-1) and Hoechst 33342 (20 μg mL-1) were added for 10 min in the dark. The cells were washed three times with PBS and observed with a Zeiss Axioplan fluorescence microscope (Zeiss), equipped with an Axiocam 503 mono microscope camera (Zeiss), excitation/emission filters 365/420 nm for blue fluorescence, and 546/590 or 565/620 nm for red fluorescence. Image acquisition and editing were done with ZEN 2 (blue edition) microscope software (Zeiss) and GNU Image Manipulation Program (GIMP 2, version 2.8.10).
AMP toxicity was determined on HKC and HDF. Keratinocytes and fibroblasts were isolated and grown in CnT-07 (CellnTEC) or R10 (Supplementary Table
10
Histological Evaluation of Bone Healing
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To evaluate the histology of healing tissue, retrieved bone specimens were fixed and decalcified before embedded in paraffin and sectioned longitudinally, which were stained with hematoxylin and eosin (H&E) and alcian blue. The histological staining was visualized and scanned using Panoptiq digital slide imaging system (ViewsIQ, Richmond, Canada).
For immunohistochemical staining, the paraffin-embedded specimen was sectioned and processed as previously described9 (link). Briefly, tissue slides were deparaffinized and rehydrated before antigen retrieval and blocking with goat serum. Then, the slides were incubated with primary antibodies (Ab), either mouse-anti-human osteocalcin Ab (ab13421, Abcam), mouse-anti-rat osteocalcin Ab (M186, Takara), rabbit-anti-rat collagen-II Ab (AB2036, Millipore) or mouse-anti-rat endothelial cells (ab9774, Abcam), before being incubated with secondary antibody conjugated with Alexa-Fluro-488 (A-11008, or A-11029, ThermoFisher Scientific). Finally, the tissue slides were covered by anti-fade mounting medium with DAPI (H-1200, Vector Laboratories) and visualized under Zeiss Axioplan fluorescence microscope (Carl Zeiss, NY).
For immunohistochemical staining, the paraffin-embedded specimen was sectioned and processed as previously described9 (link). Briefly, tissue slides were deparaffinized and rehydrated before antigen retrieval and blocking with goat serum. Then, the slides were incubated with primary antibodies (Ab), either mouse-anti-human osteocalcin Ab (ab13421, Abcam), mouse-anti-rat osteocalcin Ab (M186, Takara), rabbit-anti-rat collagen-II Ab (AB2036, Millipore) or mouse-anti-rat endothelial cells (ab9774, Abcam), before being incubated with secondary antibody conjugated with Alexa-Fluro-488 (A-11008, or A-11029, ThermoFisher Scientific). Finally, the tissue slides were covered by anti-fade mounting medium with DAPI (H-1200, Vector Laboratories) and visualized under Zeiss Axioplan fluorescence microscope (Carl Zeiss, NY).
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