4 x 105 cells per well were grown in 3D lrECM for 23 h, then treated with different levels of BEMER therapy (~13 μT and ~35 μT; 8 min) and irradiated 1 h later with 6 Gy or left unirradiated. After 24 h, cells were isolated using PBS and trypsin (PAA), fixed with 3% formaldehyde/PBS (Merck, Darmstadt, Germany), permeabilized with 0.25% Triton-X-100/PBS (Roth, Karlsruhe, Germany) and stained with specific antibodies for γH2AX and 53BP1. Samples were spread on a slide and covered with Vectashield/DAPI mounting medium. γH2AX/53BP1-positive foci were counted microscopically with an Axioscope 2 plus fluorescence microscope (Zeiss) and defined as residual DSB [34 (link)]. Immunofluorescence images were sustained using LSM 510 meta (Zeiss).
Axioscope 2 plus fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Axioscope 2 plus is a fluorescence microscope designed for versatile imaging applications. It features a high-performance optical system and enables advanced fluorescence techniques.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using axioscope 2 plus fluorescence microscope
1
3D lrECM Culture and BEMER Therapy Effects on DNA Damage
2
Cell Cycle Analysis by BrdU Incorporation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze the effect of PINCH1, ILK and ILKAP on cell cycle, the percentage of S-phase cells was determined after knockdown. Before fixation with 3% formaldehyde, cells were incubated with 10 μM BrdU for 10 min. After permeabilization with 0.25% Triton X-100/PBS for 10 min, preparation of samples with 1N HCl and 2N HCl for 10 min and blocking with 1% BSA/PBS, BrdU and Nuclei staining was accomplished with specific antibodies and Vectashield/DAPI mounting medium. Images were obtained with an Axioscope 2plus fluorescence microscope (Zeiss).
3
Quantification of Residual DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To provide further mechanistic insight into the enhanced radiosensitivity after PINCH1, ILK and ILKAP silencing, we measured residual DNA-double strand breaks (rDSB) by using the foci assay. As previously published [63 (link)], rDSBs were visualized by double staining of phosphorylated H2AX (γH2AX) plus p53 binding protein-1 (53BP1). Cells were fixed with 1% formaldehyde/PBS at 24 h after X-ray irradiation (0 or 6 Gy). Permeabilization with 0.25% Triton X-100/PBS preceded staining with specific anti-γH2AX and anti-53BP1 antibodies and Vectashield/DAPI mounting medium. γH2AX/53BP1-positive nuclear foci of at least 150 cells from three independent experiments were counted microscopically with an Axioscope 2plus fluorescence microscope (Zeiss) and defined as rDSBs.
4
Detection of DNA Double-Strand Breaks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detection of residual DNA double strand breaks (DSB), p53 binding protein-1 (p53BP1) was stained 18 (link). UTSCC15 and SAS cells were cultured under sphere-forming conditions. After 24 h, cells were irradiated with 6 Gy or left unirradiated. Two and 24 h later, cells were isolated for fixation (3% formaldehyde/PBS) and permeabilization (0.25% Triton X-100/PBS) using trypsin. After blocking with 1% BSA, cells were stained with a specific anti-p53BP1 antibody and embedded in Vectashield/DAPI mounting medium. p53BP1 positive nuclear foci were determined in at least 150 cells using an Axioscope 2 plus fluorescence microscope (Zeiss, Jena, Germany).
5
Zoospore Settlement on Coated Surfaces
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fronds of U. linza were collected from Craster, Northumberland, UK (55° 26' N; 1° 35' W) and a spore suspension of 1.0 × 10 6 spores mL -1 was prepared by the method of Callow et al. (1997) .
The experiment used 3 replicates of each coating for each treatment. All coatings were equilibrated in 0.22 μm-filtered ASW with added H 2 O 2 (0, 50, 100 and 150 µM), depending on the treatment, for 24 h prior to testing. A suspension of zoospores (10 mL; 1 × 10 6 spores mL -1 ) was added to individual compartments of quadriPERM® dishes containing the samples. After 45 min in darkness at 20 °C, the slides were washed by passing 10× through a beaker of seawater to remove unsettled (i.e. swimming) spores. Slides were fixed using 2.5% glutaraldehyde in seawater. The density of zoospores attached to the surface was counted on each of 3 replicate slides using an image analysis system attached to a Zeiss Axioscope 2 plus fluorescence microscope. Spores were visualized by autofluorescence of chlorophyll. Counts were made using Axiovision 4 software for 30 fields of view (0.15 mm 2 ) on each slide.
The experiment used 3 replicates of each coating for each treatment. All coatings were equilibrated in 0.22 μm-filtered ASW with added H 2 O 2 (0, 50, 100 and 150 µM), depending on the treatment, for 24 h prior to testing. A suspension of zoospores (10 mL; 1 × 10 6 spores mL -1 ) was added to individual compartments of quadriPERM® dishes containing the samples. After 45 min in darkness at 20 °C, the slides were washed by passing 10× through a beaker of seawater to remove unsettled (i.e. swimming) spores. Slides were fixed using 2.5% glutaraldehyde in seawater. The density of zoospores attached to the surface was counted on each of 3 replicate slides using an image analysis system attached to a Zeiss Axioscope 2 plus fluorescence microscope. Spores were visualized by autofluorescence of chlorophyll. Counts were made using Axiovision 4 software for 30 fields of view (0.15 mm 2 ) on each slide.
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!