We studied at least 10 metaphases from each specimen per method. We used Ikaros karyotyping software (Metasystems, Altlussheim, Germany) to prepare karyograms from Giemsa-stained metaphases of each species. Images were captured using a Provis AX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a DP30BW digital camera (Olympus, Tokyo, Japan) or using an Imager Z2 microscope (Zeiss, Oberkochen, Germany) equipped with a CoolCube 1 digital camera (Metasystems, Altlussheim, Germany). Photos of in situ hybridization experiments were superimposed with color and processed with DP Manager imaging software (Olympus, Tokyo, Japan) or an Isis Fluorescence Imaging System (Metasystems, Altlussheim, Germany).
Isis fluorescence imaging system
Manufactured by MetaSystems
Sourced in Germany
The ISIS fluorescence imaging system is a piece of lab equipment designed for fluorescence imaging. It provides high-resolution imaging capabilities for various applications in life science research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Axioimager 2i fluorescence microscope, by MetaSystems (3 mentions)
Donkey anti rabbit cy3 labeled antibodies, by Dianova (2 mentions)
Texas red conjugated phalloidin, by Thermo Fisher Scientific (2 mentions)
Imager z2 microscope, by Zeiss (1 mentions)
Dp30bw digital camera, by Olympus (1 mentions)
Dp manager imaging software, by Olympus (1 mentions)
Ikaros karyotyping software, by MetaSystems (1 mentions)
Provis ax70, by Olympus (1 mentions)
Rabbit anti 53bp1, by Novus Biologicals (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Fluoroshield mounting medium, by Abcam (1 mentions)
Anti ho 1, by Cell Signaling Technology (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Axioimager z2 fluorescence microscope, by Zeiss (1 mentions)
Axioscope a1 epifluorescence microscope, by Zeiss (1 mentions)
Axiocam mrm rev 3, by Zeiss (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Texas red x conjugate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti p16ink4a, by Cell Signaling Technology (1 mentions)
12 protocols using isis fluorescence imaging system
1
Karyotyping and FISH Imaging Protocols
2
Quantifying DNA Double-Strand Breaks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were taken from a healthy volunteer as indicated above. For analysis of DNA double-strand break foci we used the γ-H2AX + 53BP1 focus assay as previously described (Eberlein et al. 2015 (link); Lamkowski et al. 2014 (link)). Briefly, PBMC were isolated by Ficoll-paque (GE healthcare) density centrifugation at 1800×g for 20 min at room temperature. Cells were washed twice with PBS (pH 7,4; without Mg2+/Ca2+) and fixed in ice-cold 70% ethanol. For immunofluorescence staining (IF) we applied primary mouse anti-phospho(Ser139)-Histone H2A.X (Merck Chemicals; diluted 1:500) and rabbit anti-53BP1 (Novus Bio; diluted 1:500) antibodies and detected them with secondary goat anti-mouse Alexa-488 (Mobitec) and donkey anti-rabbit Cy3-labeled antibodies (Dianova) both at 1:1.000 dilution. Colocalization of γ-H2AX and 53BP1 foci was considered as indicative for DSB formation. Leukocyte nuclei (n = 100 per sample) were analyzed by an experienced investigator (H.S.) by manual focus enumeration using a Zeiss Axioimager 2i epifluorescence microscope equipped with a 63 × Planapochromat lens and red/green double bandpass filter (Chroma). Overlapping or deformed nuclei were excluded from the analysis. Only colocalizing γ-H2AX + 53BP1-positive foci were considered for enumeration. Images were recorded using the ISIS fluorescence imaging system (MetaSystems, Germany).
3
Centromeric FISH Probes for Aneuploidy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Centromeric Fluorescence in situ hybridization (FISH) probes (CEP) for chromosomes 7 and 15 labeled in green and for chromosomes 18 and 20 labeled in orange were used according the manufacturer's recommendations (Vysis Inc., Downers Grove, IL). FISH analyses were performed in 4–5 μm thickness sections of the TMA containing two replicates of both tumor and normal adjacent mucosa for each sample. Pretreatment included three xylene incubations, increasing concentration of ethanol series, permeabilization with EDTA and treatment with pepsin. Next, slides were incubated in 1× PBS with MgCl2 and fixed with 1% paraformaldehyde. Denaturation was performed in a Thermo Brite (Vysis) at 78 °C during 6 min for panel one (CEP7 and 18) and 85 °C during 3 min for panel two (CEP15 and 20). Hybridization was performed at 37 °C overnight. Post‐hybridization washes were performed in 0.4× SSC/0.3% NP40 at 74 °C for 2 min and 2× SSC/0.1% NP40 at room temperature during 1 min. A minimum of 100 cells were imaged with a Nikon Eclipse 50i fluorescence microscope using the Isis Fluorescence Imaging System (MetaSystems, Altlussheim, Germany). In order to infer ploidy, the weighted mean copy number of all chromosomes analyzed in each sample was calculated. A threshold for considering a highly aneuploid genome was set at 2.5, which corresponded to a hypotriploid genome (i.e., 57 chromosomes).
4
Immunocytochemistry Protocol for Visualizing HO-1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We applied immunocytochemistry as described previously (Liebau et al., 2011 (link)). Rabbit monoclonal anti-HO-1 (dilution 1:1,000, Cell Signaling, Danvers, United States) served as primary antibody before fluorescence-labeling using Alexa Fluor® 488-conjugate goat polyclonal anti-rabbit (dilution 1:500, Life Technologies, Waltham, United States). Cytoskeleton was stained using TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen, Mannheim, Germany) and nuclei were counter stained using Fluoroshield Mounting Medium (Abcam, Cambridge, United Kingdom) containing 4,6-diamidino-2-phenylindole (DAPI).
For image acquisition we used a Zeiss Axioimager 2i fluorescence microscope in combination with the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany).
For image acquisition we used a Zeiss Axioimager 2i fluorescence microscope in combination with the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany).
5
Skin Tissue Immunostaining and SMLM Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin skin tissue sections (8 μm) of the skin biopsies of day 28 post-irradiation were mounted on super-frosted slides and processed and immunostained as described in detail elsewhere [13 (link),29 (link)], with the exception that immunostaining was conducted in TCTG buffer (TRIS, 1% Na-Casein, 0.1% Tween20, 0.1% fish-gelatin, pH 7.3–7.4) to reduce background. The slides were incubated with the primary antibodies for 1 h at 37 °C in TCTG buffer followed by three 5 min washes in TCTG and incubation with the secondary antibodies for 1 h at 37 °C. The antibodies, their sources and the dilutions used are listed in Table 1 . After incubation with the secondary antibodies, sections were washed (3 × 5 min) in TCTG at 37 °C. Slides were supplied with 18 μL Prolong Gold Mounting Medium (Life technologies, Thermo Fisher Scientific, Darmstadt, Germany) containing DAPI (4′,6-diamidino-2-phenylindole) as DNA/nuclear counterstain and covered with a 24 × 60 mm cover slip. Preparations were cured for 2 days at RT and subjected to SMLM as described previously [29 (link)]. Widefield images were recorded using the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany) equipped with an Axioimager 2i and 63× lens (both Zeiss, Oberkochen, Germany).
6
Metaphase Chromosome Spread Preparation and Karyotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells in exponential growth phase were used to prepare metaphase chromosome spreads as described previously26 (link). Chromosome spreads on glass slides were stained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) to count chromosome numbers. Karyotypes were determined using the multicolour in situ hybridisation (mFISH) method. This method uses 12 painting probes, 12XCHamster (Metasystems, Altlussheim, Germany) specific for the 12 different Chinese hamster chromosomes, each labelled with different fluorochromes. Chromosome images were taken under an Axio Imager.Z2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Karyotypes were analysed using the Isis fluorescence imaging system (Metasystems).
7
Telomeric Mapping: PNA Probe Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Telomeric mapping was performed as described previously [27 (link)]. Briefly, 10 μl of hybridisation mixture containing 70% formamide, 0.3 μg/ml Cy3-(CCCTAA)3 peptide nucleic acid (PNA) probe (Biosynthesis, Inc., Texas) and 1× Denhardt’s solution in 10 mM Tris pH 7.2 were added to the slide under a coverslip and sealed with rubber cement. The DNA was denatured by heating for 3 min at 80°C. After hybridisation for 2 h at 37°C in a humidified chamber, the slides were washed at room temperature with 70% formamide, 1% BSA, 10 mM Tris pH 7.2 (2 times for 15 min) and then with 0.1 M Tris, 0.15 M NaCl, pH 7.5 containing 0.08% Tween-20 (3 times for 5 min). The slides were then dehydrated through an ethanol series (1 min in each of a 70%, 90% and 100% solution), air dried, stained with DAPI (4′, 6′-diamidino-2-phenylindole) (50 μg/ml DAPI solution in 2 × SSC) for 45 s at room temperature and mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA). Images were captured using a Zeiss Axio Scope A1 epifluorescence microscope fitted with a high-resolution microscopy camera AxioCam MRm Rev. 3 (Carl Zeiss Ltd.) and analysed using AxioVision v4.8.1 software or ISIS Fluorescence Imaging System (MetaSystems, Altlussheim, Germany).
8
Immunofluorescence Staining of hnRNP K and p53
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The IF staining was performed as previously described (13 (link)) using a rabbit monoclonal antibody targeting hnRNP K (1:250, Biozol; cat. no. LS-C30312-50)/mouse monoclonal antibody targeting Ser15-phospho-p53 (1:250, Cell Signaling Technology, Inc.; cat. no. 9286). Fluorescence-labeled secondary antibody (Alexa Fluor® 488-conjugate; donkey polyclonal anti-rabbit, 1:500; Thermo Fisher Scientific, Inc.; cat. no. A-21206) and Texas-Red-X-conjugate (goat polyclonal anti-mouse, 1:500; Thermo Fisher Scientific, Inc.; cat. no. T-6390). Primary and secondary antibodies were incubated for 1 h at room temperature in the dark. For image acquisition, a Zeiss Axioimager 2i fluorescence microscope and the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany) were used.
9
Immunofluorescence Staining of HNSCC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IF microscopy was performed as previously described (26 (link)). For IF staining of HNSCC cells, TexasRed-conjugated Phalloidin (dilution 1:40, Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-hnRNP K (dilution 1:1,000, Cell Signaling Technology, Inc.) and a rabbit polyclonal anti-p16INK4A (dilution 1:1,000, Cell Signaling Technology, Inc.) were used following 60 min of incubation at RT. The nuclei were stained using Fluoroshield Mounting Medium with DAPI (Abcam). Fluorescence labeling was carried out with Alexa Fluor® 488-conjugated goat polyclonal anti-mouse and Alexa Fluor® 488-conjugated goat polyclonal anti-rabbit antibodies (both from Thermo Fisher Scientific, Inc.; dilution 1:500). Image acquisition was performed with a Zeiss AxioImager 2i fluorescence microscope (Carl Zeiss AG) and the ISIS fluorescence imaging system (MetaSystems).
10
Fluorescence Imaging with Zeiss Microscope
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescent images were recorded using the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany) containing a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a motorized filter set for excitation of immunofluorescence in red, green and blue and 40× and 63× Plan-Neofluar oil immersion lenses (Zeiss, Jena, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!