Glial fibrillary acidic protein (gfap)

Following cardiac perfusion with cold phosphate buffered saline (Fisher Scientific), brain and tumor tissues were dissected and fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned at 5 μm thickness. Tumor tissue sections were deparaffinized, rehydrated and stained using standard immunohistochemistry and immunofluorescence protocols, as described before [26 (link), 32 (link), 39 (link)]. The following antibodies were used: phospho-Erk (Thr202/Tyr204), Ki67/Mib-1, Met, phospho-Met (Tyr1234/1235), Pten, phospho-S6 ribosomal protein (Ser235/236) (Cell Signaling Technology), Gfap (Biocare Medical), Olig2, Sox2 (Abcam), Tuj1 (Sigma), 53BP1 (Santa Cruz Biotechnology), horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories), and Alexa488/568-conjugated secondary antibodies (Thermo Fisher). For immunofluorescence, 4’,6-diamidino-2-phenylindole counterstain (DAPI, Vector Labs) was used to visualize nuclei. Ki67 indices were quantified by manually counting at least 500 nuclei in the area of highest staining, as described [40 (link)]. All immunohistochemistry images were acquired on a Keyence BZ-X800 microscope using the Keyence BZ-X software (Keyence). All immunofluorescence images were acquired on a Zeiss Axio Imager 2 microscope using the Zen Imaging software (Carl Zeiss).

Formalin-fixed, paraffin-embedded brain sections were deparaffinized, re-hydrated, and stained using standard protocols, as described previously (30 (link)). Briefly, antigen retrieval was performed at 98°C for 20 min, at 15psi, in 10mM citrate buffer (pH 6) in a commercial pressure cooker for all antibodies. Tissue sections were blocked in Goat Serum (Vector Laboratories) for 30 minutes in TBS prior to overnight incubation in primary antibody, following which sections were incubated with Alexa 488/568- conjugated secondary antibodies (Invitrogen) for 1 hour prior to mounting in Vectashield with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories). The following primary antibodies were used: p21 (Abcam, Cat. No. 1888224), Lamin B1 (Abcam, Cat. No. 16048), pMET Tyr1234/1235 (Cell Signaling, Cat. No. 3077S), GFAP (Biocare, Cat. No. CP040A). Immunofluorescence images were acquired with a Zeiss Axio Imager 2 motorized inverted microscope with an Axiocam camera and ZEN imaging software (Carl Zeiss). The percentage of p21+/GFAP+, Lamin B1+/GFAP+, or Lamin B1+/DAPI+ cells was calculated by counting the number of GFAP− or DAPI-positive cells that also co-stained with p21 or Lamin B1 per visual field (40X).