Decreasing in vivo and in vitro phospho-signals as a consequence of slow formalin diffusion.

(A) Glioblastoma tissue was processed for routine diagnostics, and in addition, a primary spheroid culture from the same tissue was generated. The spheroid culture was spun down and supernatants were aspirated. We slowly added 4% formalin to the 15 ml tube to avoid a resuspension of cells. The fixated pellet was cut at the upper side as well as in the middle of the conus. Finally, the material was processed for paraffin-embedding according to the standard diagnostic procedure. (B) Macroscopic overview of 4 stained sections with cell pellet and corresponding glioma specimen. (C) Detection of phospho-RPS6 (Ser235/236), phospho-RPS6 (Ser240/244) and phospho-4EBP1 (Thr37/46) in glioblastoma tissue (large image tumor border, small inlay image tumor center). (D) Phospho-stainings in the corresponding spheroid culture according to different cutting levels within the cell pellet. (E) Longitudinal section through a LNT-229 FFPE glioma cell pellet showing a strong gradient from superficial cell layers to the bottom of the pellet.