ActinRed™ 555 ReadyProbes™ reagent, NucBlue™ Fixed Cell Stain ReadyProbes™ reagent, and an Image-it™ Fixation/Permeabilization kit (Life Technologies Corp., Eugene, OR, USA) were employed in this study to investigate the condition of the cellular structure when subjected to the 5-FU loaded film solution. A single round (22 mm) collagen-coated coverslip (BioCoat, Bedford, MA, USA) was placed into each well of a 6-well plate for the cells to adhere to. The seeding density of the cells was 0.3 × 106 per well. The total volume of cells and media was 3 mL for each well. Cells were allowed 48 h within an incubator to reach confluency. Control wells where the cell line was not treated did not contain EPS/5-FU samples. Exposure experiments saw the addition of a 100 µL of EPS/5-FU film-forming solution added to the well. At 12 and 36 h after exposure, cells were immediately fixed using 4% formaldehyde in PBS. Once this was achieved, either the actin filaments or nucleus of the cells were marked for analysis with the utilization of epifluorescent stain. All the acquired images were not manipulated or altered prior to measuring corrected total cell fluorescence (CTCF). Photographs for both stains used the same imaging settings for that respective group. Images were viewed and photographed using an Olympus IX50 inverted fluorescence microscope.
Image it fixation permeabilization kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Image-iT Fixation/Permeabilization Kit is a laboratory reagent designed for the fixation and permeabilization of cells prior to immunofluorescence staining and analysis. The kit contains the necessary components to prepare samples for intracellular staining and visualization using fluorescence microscopy or flow cytometry.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Axiovert microscope, by Zeiss (2 mentions)
Actin green probes, by Thermo Fisher Scientific (2 mentions)
Black walled 96 well imaging microplate, by Corning (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Actinred 555 readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Nucblue fixed cell stain readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Ix50 inverted fluorescence microscope, by Olympus (1 mentions)
Slowfade gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Axiovison software v4, by Zeiss (1 mentions)
Plan apochromat x63 n a 1, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Slowfade diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Fluor 60 water immersion objective, by Nikon (1 mentions)
24 protocols using image it fixation permeabilization kit
1
Evaluating 5-FU Film Effects on Cell Structure
2
Actin Cytoskeleton Imaging of SMCs
3
Immunostaining of Apoptosis Markers
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Cells were allowed to adhere to polylysine-L slides (Thermo Scientific) and analyzed using an Image-iT® Fixation/Permeabilization Kit (Life Technologies). Briefly, cells were fixed for 15 minutes with 4% paraformaldehyde in PBS (pH7.3) at room temperature, then permeabilized with Triton X-100 0.5% for 15 minutes and blocked for 1 hour with 3% BSA in DPBS (pH7.4). Cells were incubated with cleaved caspase-3 antibody (rabbit monoclonal Ab 9664; Cell Signaling Technology) or cytochrome c antibody (mouse monoclonal Ab 556432; Becton Dickinson) overnight at 4°C. Secondary antibodies were goat anti-rabbit and goat anti-mouse IgG coupled with Alexa 568 (red) or Alexa 488 (green) fluorochromes (Life Technologies) incubated at 37°C for 2h. Nuclei were counterstained with DAPI (Vector). Images were acquired by immunofluorescence microscopy on a Zeiss Axiovert microscope with a Plan-Apochromat X63 N.A.1.4 oil immersion objective using the Axiovison software v4.2 (Carl Zeiss).
4
Cytoskeletal Dynamics in Coralyne-Treated Cells
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Cells were seeded onto sterile round coverslips coated with poly-L-lysine (Thermo Scientific, US), treated with 25 µM coralyne and exposed to IR according to the scheme of the experimental procedure (Figure 11 ). Thereafter, cells were fixed using an Image-iT® Fixation/Permeabilization Kit (Life Technologies, US). Briefly, cells were fixed for 15 min with PBS (pH 7.3) containing 4% paraformaldehyde at room temperature, permeabilized with 0.5% Triton X-100 for 15 min, and blocked for 1 h with Dulbecco’s PBS (pH 7.4) containing 3% bovine serum albumin (BSA). The organization of the F-actin network was evaluated in cells stained with rhodamine phalloidin (Invitrogen, US) diluted 1:300 with 0.1% BSA for 20 min at room temperature. The organization of the integrin-β1 network was analyzed in cells stained with an anti-integrin-β1 antibody conjugated with FITC (Abcam, Cambridge, UK) diluted 1:100 with 0.1% BSA for 60 min at room temperature. After staining, cells were washed three times with PBS for 10 min and mounted with SlowFade™ Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA, USA) to visualize nuclei. Fluorescent signals were observed with a confocal laser scanning microscope (Nikon A1) equipped with a Nikon Fluor ×60 water immersion objective.
5
Astrocytic GFAP Expression Analysis
6
Immunofluorescence Analysis of Cellular Cytoskeleton
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HFL1 cells were seeded in count 40,000 per well on the bottom of 12-well plate and treated as described above.
For further processing, Image-iT® Fixation/Permeabilization Kit (Life Technologies, Prague, CZ) was used. Samples were fixed with 4 % formaldehyde for 15 min, permeabilized in Permeabilization solution for 15 min and blocked in 3 % BSA for 60 min. Cells were then sequentially incubated overnight with the primary monoclonal Anti-Vinculin (V9131) and monoclonal Anti-Vimentin (V5255) antibodies. Secondary Anti-Mouse IgG-Atto 488 goat antibody (62197) and Anti-Mouse IgM-FITC goat antibody (F9259) were applied for 60 min at room temperature in dark. For actin staining, Phalloidin-Atto 488 (49409) was used. Samples were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen, Life Technologies, Prague, CZ) and analyzed using the Olympus IX81 fluorescent microscope (Olympus; Tokyo, Japan) equipped with a Cell-R system at 40×, 100×, and 400× magnification.
Cellular mitochondrial network was visualized with MitoTracker™ Red CMXRos (No. M7512; Thermo Fisher Scientific, Prague, CZ) using 0.5 µl of 1 mmol/l stain for each well containing 1 ml of the cell culture medium. Basic evaluation was followed by fluorescent microscopy at a 100× magnification by the Hamamatsu Orca-ER camera mounted on the Olympus IX 81 inverted microscope (Olympus; Tokyo, Japan).
For further processing, Image-iT® Fixation/Permeabilization Kit (Life Technologies, Prague, CZ) was used. Samples were fixed with 4 % formaldehyde for 15 min, permeabilized in Permeabilization solution for 15 min and blocked in 3 % BSA for 60 min. Cells were then sequentially incubated overnight with the primary monoclonal Anti-Vinculin (V9131) and monoclonal Anti-Vimentin (V5255) antibodies. Secondary Anti-Mouse IgG-Atto 488 goat antibody (62197) and Anti-Mouse IgM-FITC goat antibody (F9259) were applied for 60 min at room temperature in dark. For actin staining, Phalloidin-Atto 488 (49409) was used. Samples were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen, Life Technologies, Prague, CZ) and analyzed using the Olympus IX81 fluorescent microscope (Olympus; Tokyo, Japan) equipped with a Cell-R system at 40×, 100×, and 400× magnification.
Cellular mitochondrial network was visualized with MitoTracker™ Red CMXRos (No. M7512; Thermo Fisher Scientific, Prague, CZ) using 0.5 µl of 1 mmol/l stain for each well containing 1 ml of the cell culture medium. Basic evaluation was followed by fluorescent microscopy at a 100× magnification by the Hamamatsu Orca-ER camera mounted on the Olympus IX 81 inverted microscope (Olympus; Tokyo, Japan).
7
Immunofluorescence Imaging of Cells
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Cells were fixed and permeabilized with Image-iT® Fixation/Permeabilization Kit (Thermo Fisher Scientific) and were blocked in blocking buffer (0.1 M glycine, 2% BSA, 0.1% Triton-X100 in PBS). Primary antibodies were diluted in blocking buffer and incubated at 4 °C overnight. After washing, samples were incubated with alexa488-, alexa568- and alexa647-conjugated secondary antibodies for 2 h at room temperature and thereafter mounted using Dako mounting media. Samples were captured using an SP8 confocal system (Leica, Wetzlar, Germany) and image analysis was performed using Image J (NIH, USA)
8
Quantifying Micronuclei in Cancer Cells
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HCC15 CTNNB1Δ cells expressing vector alone or CTNNB1 variants were plated in a black walled 96-well imaging microplate (Corning, NY) at a density of 1,000 cells/well. Mock and irradiated (6 Gy) cells were fixed and permeabilized 72 hours post treatment using the Image-iT Fixation/Permeabilization Kit (Thermo Fisher Scientific). After fixation, the cytosol and nuclei were stained using actin green probes (green; ThermoFisher, MA) and DAPI (blue; ThermoFisher, MA), respectively. For each cell line, at least 4 wells with 16 images per well (64 images in total) were captured at 40X magnification using a Cytation 5 cell imaging multimode reader (BioTek, VT). All images were processed manually using the ImageJ software. We applied an ellipsoid fit and used size and circularity parameters based on the DAPI intensity to quantify micronuclei (24 ).
9
Quantifying Micronuclei in HCC15 Cells
10
Immunodetection of BCR-ABL Fusion Protein
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We generated an anti‐BCR‐ABL (e14a2) junction‐specific Ab for immunocytochemistry by immunizing rabbits with the peptide C + KQSSVPTSSKENLL corresponding to amino acids 78‐91 (EU394718.1) of the e14a2‐type BCR‐ABL protein (Figure S1 ). Specificity of the antibody was validated by peptide blocking and negative staining of e1a2‐type BCR‐ABL protein in Ph‐positive ALL cells (Figure S2 ). For immunofluorescent staining, cells were placed on a glass slide using a Cytospin centrifuge (Shandon Scientific) or cultured on a chamber slide and fixed using an Image‐iT Fixation/Permeabilization Kit (Thermo Fisher Scientific).24 These specimens were stained with a rabbit BCR‐ABL junction‐specific Ab and anti‐p62 (MBL), anti‐CD34, or anti‐pan 14‐3‐3 (Santa Cruz Biotechnology) Abs. We used Alexa Fluor 594‐conjugated anti‐rabbit IgG and Alexa Fluor 488‐conjugated anti‐mouse IgG Abs (Invitrogen) as secondary Abs. Finally, nuclei were counterstained with ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology). Stained samples were observed under a BZ‐X fluorescence microscope (Keyence).
Other conventional techniques and reagents are described in DocumentS1 .
Other conventional techniques and reagents are described in Document
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