Biotinylated rabbit anti mouse

Staining of human tissues was performed as described (31). The paraffin embedded sections were cleared and the sections were incubated with 0.1% Pronase (Roche #165 921) in 0.1% CaCl2 pH 7.8. at 37C for 10 minutes. They were blocked with 3% H2O2 in TBS for 10 mins., washed then blocked with Dako Biotin Blocking System (Dako X0590). After washing, they were further blocked with 10% Normal Rabbit Serum for 10 mins at room temperature (RT) and incubated firstly with p-ERK (Abcam, ab65142) dilution 1:50, PKM2 (Abcam, ab38237) dilution 1:100 and GLUT-1 (Abcam, ab652) dilution 1: 100for 1hr at RT, then with biotinylated Rabbit Anti-Mouse (Dako, E-0354) at 1/100 for 30 mins. at RT, and finally with Strep-ABC complex (Dako, K-0377) at 1/100 for 30 mins. at RT. The sections were developed with AEC substrate kit (vector lab, SK-4200) at RT for 20 mins., counterstained with haematoxylin and mounted with DAKO aqueous mount (Dako, 003181).

Immunohistochemistry using sections of paraffin-imbedded tumors was carried out as previously described [30] (link). Briefly, after antigen retrieval (microwave, citrate buffer, ph = 6), mouse anti-YKL-40 or corresponding isotype control (see above) was used as first antibody, respectively, followed by incubation with biotinylated rabbit anti-mouse (Dako, Glostrup, Denmark) and visualization using the streptavidin-alkaline-phosphatase based Vectastain ABC kit (Vector, Burlingame, CA, USA). Slides were scanned by a Mirax microscope (Zeiss, Jena Germany) and the Mirax Viewer (Zeiss) software was used to take images. Other antibodies used for immunohistochemistry (with corresponding biotinylated secondary antibodies (all Dako)) were: Anti murine EDG-1 (S1P1) (Santa Cruz, Heidelberg, Germany), anti-murine CD45 (clone 30-F11, BD) and anti-human Ki-67 (clone MIB-1, Dako). Images of four randomly chosen fields of vision (not containing necrotic areas) were taken with the viewer software. Human Ki-67 or murine CD45 positive cells were counted and murine S1P1 positive areas quantified using the ImageJ software (Wayne Rasband, NIH, USA). Positive cells or percent positive area per field of vision were calculated for each tumor.
Histochemical Masson-Goldner staining of paraffin-imbedded tumors was performed as described [33] .

Western blot was carried out as previously described49 (link). The primary antibodies used were mouse monoclonal anti-glutamine synthetase (Abcam, Cambridge, UK), anti-β-actin antibody (Sigma-Aldrich, Saint-Louis, Missouri, USA), anti-α-tubulin antibody (Santa Cruz, Dallas, Texas USA.), rabbit polyclonal anti-asparagine synthetase (Abcam), anti-4E-BP1, anti-4E-BP1-pT70, anti-Erk1/2, anti-Erk1/2-pT202/Y204, anti-elF-2α-pS51, anti-elF-2α (Ozyme, Saint Quentin Yvelines, France), and anti-BCL2A1 antibody (Abcam). Secondary antibodies were as follows: biotinylated rabbit-anti mouse (Dako, Courtaboeuf, France) and biotinylated goat anti-rabbit (Invitrogen, Carlsbad, CA, USA). Detection was performed using Luminata Forte Western HRP substrate (Millipore Corporation, Billerica, MA). Quantifications were carried out by densitometric analysis using Kodak software as previously described49 (link). β-actin or α-tubulin was used as loading control.