Figure 6.
Confocal immunofluorescence microscopy of wt-TGH and KDEL-TGH. (A) McArdle RH7777 cells stably expressing flag-tagged wt-TGH and myc-tagged KDEL-TGH were fixed onto microscope coverslips and incubated with a monoclonal mouse anti-flag antibody and polyclonal rabbit anti-myc antibodies as described in Materials and Methods. The flag-tagged wt-TGH and myc-tagged KDEL-TGH were visualized with secondary anti-mouse and anti-rabbit antibodies conjugated to Texas Red and fluorescein or Alexa488, respectively. Images were obtained using a confocal microscope. The boxed region is shown under higher magnification. Bars, 2 μm for higher magnification (left); 5 μm for lower magnification (right). (B) McArdle RH7777 cells stably expressing wt-TGH or KDEL-TGH without epitope tags were fixed onto microscope cover slips as in A. Incubations were performed with a monoclonal mouse anti-BiP antibody and polyclonal rabbit anti-TGH antibodies, which were stained with secondary anti-mouse and anti-rabbit antibodies conjugated to fluorescein and Texas Red, respectively. Images were obtained on a confocal microscope as they were in A.