Anti met

Ears were collected, fixed in 4% PFA at 4°C overnight and washed. Then, they were cryoprotected in 30% and embedded in Tissue-Tek OCT (Sakura). 20- or 8-μm thick OCT sections were thawed for 30 minutes at room temperature, fixed in 4% PFA for 10 minutes and permeabilized with 0.3% Triton X-100 in PBS. After 30 minutes in blocking buffer (0.5% BSA, 5% donkey serum, 0.3% Triton X-100, 0,1% NaN3), slides were incubated with primary antibodies overnight at 4°C. The next day slides were washed with 0.3% Triton X-100 in PBS for 30 minutes and appropriate alexa secondary antibodies 488/555 were added 1:500 in blocking buffer for 1 hour at RT. After that, slides were washed with 0.3% Triton X-100 in PBS for 30 minutes and mounted.
The following antibodies were used: either unlabelled or biotin-labelled Ly6G (Biolegend), anti-F4/80-APC (Invitrogen), anti-MET (Abcam), anti-Phospho-MET (Tyr1234/1235) (Cell Signaling Technology), anti-CD206 (Biolegend). Images were acquired on a Zeiss LSM880 and analysed using Imaris. Quantification of Ly6G⁺ and F4/80⁺ cells was performed using ImageJ software. 3D rendering and visualisation were also generated using the Imaris cell imaging software.

The procedure of fixation and antibodies staining for immunofluorescence and immunohistochemistry were performed as described [61 (link)]. For the immunofluorescence analysis, the images were acquired using Zeiss Apotome fluorescence microscope and AxioVision Rel. 4.7 software. (Carl Zeiss, Gottingen, Germany). The following primary antibodies were used at the recommended dilutions: anti-Flag (Sigma-Aldrich, F3165), anti-MET (Abcam, ab51067) and anti-AKT (Cell signaling technology, SC-5298). Images were acquired using a Carl Zeiss Axiovert 200 M microscope and AxioVision Rel. 4.7 software. For immunohistochemistry staining, ABclonal IFITM3 Rabbit pAb ABclonal Inc., USA, was used at the recommended dilutions. The avidin–biotin complex method and scoring formula were employed according to the previously described method [62 (link)]. The IHC staining intensities were graded as absent (0), weak (1 +), medium (2 +) and strong (3 +). The histoscore (Q) was calculated with multiplying the percentage (P) of positive GC cells by the intensity (I) according to the formula: Q = P1 x I1 + P2 x I2 + Pn x In.

A431 tumors were fixated in formalin directly after removal. Next, tumors were paraffin-embedded, sectioned, and deparaffinised. Antigen retrieval was achieved by microwaving (10 + 15 min) in citrate buffer (DAKO, S2369) or Tris-EDTA buffer (DAKO, S2367). Immunostaining with anti-HSP90 (Abcam, UK), anti-DNA-PKcs (Abcam, UK), anti-CD44 (Abcam, UK), anti-CD44v6 (AbD serotec), anti-EGFR, anti-MET and anti-ATM (Abcam, UK) performed according to manufactures instructions. The secondary step has been Dako EnVision+ System-HRP labeled polymer anti-rabbit or EnVision FLEX/HRP (K8000, Dako). The sections were counterstained with Mayer's hematoxylin (DAKO).