Visualization of isolated subsets of stem EpCAM-negative CTCs was carried out on confocal microscope LSM 780 NLO (Carl Zeiss, On Cohen, Germany). In order to do that cell suspension after sorting was dried on glass with poly-l -lysin coating, fixated with cold methanol and incubated with 3% BSA in 1× PBS with 0.02% Tween 20 (Amresco, Dallas, TX, USA) during 45 min for preventing non-specific antibody binding. Further the primary antibodies rabbit anti-EpCAM (polyclonal, 1:2000, Abcam, Cambridge, UK) and goatanti-CK7 (polyclonal, 1:50, Santa Cruz Biotechnology, Dallas, TX, USA) in 1% BSA were added and incubated in dark during 30 min. Then were washed with PBS and after that the cocktail of second conjugated antibodies Anti-Rabbit IgG H&L (Cy3) (Abcam, Cambridge, UK) and Anti-Goat IgG H&L (AlexaFluor 647) (Abcam, Cambridge, UK) was added. Sections were covered by Mounting Medium with DAPI (Dako, Carpinteria, CA, USA) and analyzed with the laser scanning microscope LSM 780 NLO (Carl Zeiss, Oberkochen, Germany) with magnification ×630.
Mounting medium with dapi
Manufactured by Agilent Technologies
Sourced in United States
Mounting Medium with DAPI is a laboratory product used to prepare samples for microscopic analysis. It is a liquid solution that helps secure and preserve biological specimens on microscope slides. The product contains DAPI (4',6-diamidino-2-phenylindole), a fluorescent dye that binds to DNA, allowing for the visualization of cell nuclei.
Automatically generated - may contain errors
3 protocols using mounting medium with dapi
1
Visualizing Isolated EpCAM-Negative CTCs
2
Imaging of γδ T Cell Activation
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γδ T cells or Vγ4+ cells were flow purified using a FACSAria Fusion (BD Biosciences). Purified cells were transferred onto poly-L-lysine–coated chamber slides, incubated for 2 h at 37°C, fixed in 4% paraformaldehyde for 15 min, and blocked with 20% FCS for 20 min γδ T cells were immunostained with anti-TCRβ–Alexa Fluor 647 and phalloidin, and Vγ4+ cells were immunostained in 5% bovine serum albumin with rabbit anti-TCRβ, washed, and labeled with a goat anti-mouse secondary antibody conjugated to Alexa Fluor 594. Slides were mounted using Mounting Medium with DAPI (DakoCytomation) and viewed on a point-scanning confocal microscope (FV1000; Olympus). Confocal images were selected to represent at least 20 captures (n = 3 independent experiments).
3
Fluorescent Staining of Adherent Cells
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Slides were plated on coverslips and allowed to adhere for 24 h. After adherence, cells were treated with drug for 24 h. After treatment, the drug was removed, and cells were washed once in PBS. Cells were labeled with a 50 mM concentration of autofluorescent marker MDC (Sigma) in PBS for 10 min at 37°C. Cells were fixed in 4% formaldehyde for 15 min at room temperature. Cells were washed twice in PBS for 5 min and mounted on slides using Dako mounting medium with DAPI. Coverslips were sealed with clear nail polish and imaged with a 3DHistech MIDI scanner as described above.
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