Mab1976

Immunofluorescence staining was performed as previously described [29 (link)]. Cells were fixed with 4% of paraformaldehyde and were specified and permeabilized for 15 min using 0.05%Triton X-100/PBS. Subsequently, cells were blocked using 5% w/v bovine serum albumin (BSA) in PBS for 1h at room temperature. Localization studies were performed using primary antibody to HIF1-α (Cell signaling technology; #3434, 1:50), integrin αvβ3 (MILLIPORE; MAB1976, 1:100), LIF (NovusBiologicals; NBP1-85717, 1:100), CD31 (abcam; ab28364, 1:100), and CD34 (abcam; ab8536, 1:100) and further incubated with anti-rabbit IgG fluorescence (Invitrogen) or anti-mouse IgG fluorescence (Invitrogen) as appropriate, all at 1:400 for 1h. Cover glasses were mounted in Vectashield mountant with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) as nuclear stain. For the negative control, cells were incubated under similar conditions with isotype-matched mouse or rabbit immunoglobulin in the place of the primary antibodies. Images were captured using oil immersion 63x objectives Zeiss 510 microscopy (Carl Zeiss MicroImaging, Röttingen, Germany) and processed using Zen software (ZEISS).