For immunofluorescence, cells in 3D culture were stained using a protocol modified for sandwich Matrigel [11 (link), 20 ]. The samples were first rinsed by PBS—glycine (100 mM glycine in PBS) three times, then blocked with 10% goat serum (Sigma), 1% goat F(ab′)2 anti-mouse immunoglobulin G (Caltag, UK) in staining buffer (PBS supplemented with 0.2% TritonX-100, 0.1% BSA, and 0.05% Tween 20). The samples were then incubated with the desired primary antibodies at 4 °C overnight (Tuj1 rabbit anti-human (Abcam, UK), SOX-2 mouse anti-human (Merck Millipore, UK)), followed by the incubation in the suitable second antibodies (Goat anti-rabbit Alexa569, goat anti-mouse Alexa488 from ThermoFisher, UK) at room temperature for 45 min at least and finally were mounted with diaminophenylindole (DAPI) mounting medium (Abcam, UK). Images were taken on a confocal microscope (Zeiss 710), viewed in ZEN software (Zeiss) and exported into ImageJ (http://imagej.nih.gov/ij/ ) for processing.
Diaminophenylindole dapi mounting medium
Manufactured by Abcam
Sourced in United Kingdom
Diaminophenylindole (DAPI) mounting medium is a fluorescent dye used to stain and visualize DNA in fixed cells or tissue samples. It binds to the minor groove of DNA, emitting a blue fluorescence when excited by ultraviolet light. This medium is commonly used in fluorescence microscopy applications to label cell nuclei.
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2 protocols using diaminophenylindole dapi mounting medium
1
Immunofluorescence of 3D Cell Cultures
2
Immunofluorescence Staining of ReN VM Spheroids
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ReN VM spheroids in matrix were fixed by 4% PFA for at least 15min and stained using a protocol modified from previous studies. The samples were first washed using PBS -glycine (100 mM glycine in PBS) three times, then blocking buffer (10% goat serum (Sigma), 1% goat F (ab')2 anti-mouse immunoglobulin G (ThermoFisher, A24514) in staining buffer (PBS supplemented with 0.2% TritonX-100, 0.1% BSA, and 0.05% Tween 20) was added. The samples were then incubated with primary antibodies at 4 °C overnight or at room temperature for 1.5 hours, followed by incubation in suitable second antibodies (Human anti-rat Alexa 555 A21434, human anti-rabbit Alexa 488 A27034) at room temperature for 45 min and finally were mounted with diaminophenylindole (DAPI) mounting medium (Abcam, UK). Images were taken and processed on a confocal microscope (Nikon, UK).
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