Colibri light source

Tumorspheres were fixed free-floating in methanol for five minutes before embedding in paraffin for sectioning. 5 µm sections of embedded spheroids were cut on a Leica RM 2255 microtome (Nussloch, DE) and fixated on glass slides by melting of paraffin residue at 60°C for one hour. Sections were stained using primary antibodies Rb anti-GFAP (Dako; #Z0334; 1:200) and Ms anti-human nestin (Abcam; #ab22035; 1:200). Secondary antibodies were Dnk-anti-Ms-Alexa-488 (Invitrogen; #A-21202; 1:500) and Gt-anti-Rb-Alexa-594 (Invitrogen, CA, USA; #A-11037; 1:500). Antigen retrieval was performed using a 10 mM sodium citrate buffer (pH 6.0) with 0.05% Tween. Cells were additionally immunostained with 4,6-diamidino-2phenylindole (DAPI) (Sigma; #000000010236276001; 1:500) for nuclear staining. Slides were mounted using fluorescent mounting medium (Dako; #S3023) and images were acquired on a Zeiss Observer Z.1 using the Colibri light source (Zeiss) and Orca-Flash4.0 V2 (Hamamatsu) as the detector. To quantify the area that nestin and GFAP signal covers, the manual threshold tool in Fiji was used. Five images of five different tumorspheres were used for thresholding and the area coverage in percent was normalized for each tumorsphere to the respective tumorsphere size determined by area of the nuclear stain (DAPI) when oversaturated. Data was plotted and analyzed in GraphPad Prism 6.