Timeline of in vivo and in vitro assessment of phospho-signals before formalin-fixation.

(A) In vitro detection of phospho-RPS6 (Ser235/236), phospho-RPS6 (Ser240/244) and phospho-4EBP1 (Thr37/46) 0–60 minutes after harvesting LNT-229 glioma cells. Cells were kept at room temperature without medium in an open 15 ml plastic tube. Considerable reduction of p4EBP1 (Thr37/46) signal intensity was observed after 60 minutes. (B) In vivo detection of phospho-signals 35–230 minutes after surgical resection of a GBM. As a control to show general immunogenicity of the tissue samples we used an antibody against GFAP. While staining signals of phospho-RPS6 (Ser235/236) and phospho-RPS6 (Ser240/244) did not show considerable decrease over the respective time intervals, we hardly detected any signal for phospho-4EBP1 (Thr37/46) in the tissue specimens.