Isolectin b4 af568

Excised NGP tumors were embedded in Tissue-Tek® optimum cutting temperature (O.C.T.) compound (Electron Microscopy Services), then stored at -20 ºC until cryosectioned (Leica CM1860). 15 μm thick cryosections were fixed with acetone and permeabilized with Tween 20. After blocking non-specific binding with CAS-Block Histochemical Reagent (ThermoFisher Scientific), the following primary antibodies were used: murine iNOS (1:500, #13120, Cell Signaling), aSMA-Cy3 (1:1000, #C6198, Sigma), and pimonidazole (1:100, #Pab2627, Omnikit, Hypoxyprobe, Massachusetts). Isolectin-B4-AF568 (1:100, #I21412, Invitrogen) was diluted in HEPES buffer. Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) secondary antibody was applied following incubation in primary solution and a series of washing steps. Finally, the slides were mounted with DAPI (VECTASHIELD PLUS Antifade Mounting Medium with DAPI, Vector Laboratories).

Excised NGP tumors were embedded in Tissue-Tek® optimum cutting temperature (O.C.T.) compound (Electron Microscopy Services), then stored at -20 ºC until cryosectioned (Leica CM1860). 15 μm thick cryosections were fixed with acetone and permeabilized with Tween 20. After blocking non-specific binding with CAS-Block Histochemical Reagent (ThermoFisher Scientific), the following primary antibodies were used: murine iNOS (1:500, #13120, Cell Signaling), aSMA-Cy3 (1:1000, #C6198, Sigma), and pimonidazole (1:100, #Pab2627, Omnikit, Hypoxyprobe, Massachusetts). Isolectin-B4-AF568 (1:100, #I21412, Invitrogen) was diluted in HEPES buffer. Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) secondary antibody was applied following incubation in primary solution and a series of washing steps. Finally, the slides were mounted with DAPI (VECTASHIELD PLUS Antifade Mounting Medium with DAPI, Vector Laboratories).