Figure 3.
Confocal and STED microscopy reveals that internalized SSTR2 localizes to a cellular compartment distinct from the TGN. (A) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for each treatment condition. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. (B) AtT20 cells were fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for each set of images. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Dotted lines indicate the plasma membrane. (C) AtT20 cells were incubated for 3 min with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SST2R. Images were collected using standard confocal microscopy, and the area under the magnifying glass was acquired by STED microscopy. Scale bars represent 5 µm and 1 µm for the confocal and STED images, respectively. Dashed areas in the STED image indicate individual SSTR2-labeled vesicles. (D and E) AtT20 cells were incubated for 40 min with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SSTR2 (D) or syntaxin-6 (E). Images were collected using standard confocal microscopy and the area under the magnifying glass was acquired by STED microscopy. Scale bars represent 5 µm and 1 µm for the confocal and STED images, respectively. Dashed lines in the confocal image indicate the cell boundary and dashed lines in the STED image indicate tubulovesicular structures. (F) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for the [D-Trp8]-SOM treatment condition. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Images were acquired using an Abberior STED microscope as a single optical section through the cells and cropped using ImageJ. Dashed lines indicate cell boundaries. (G) AtT20 cells were fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification in the bottom row for each image. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Images were acquired using an Abberior STED microscope as a single optical section through the cells and cropped using ImageJ. Dashed lines indicate cell boundaries. The arrowheads indicate the spatial segregation of immunoreactive signals of the indicated proteins.

Confocal and STED microscopy reveals that internalized SSTR2 localizes to a cellular compartment distinct from the TGN. (A) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for each treatment condition. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. (B) AtT20 cells were fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for each set of images. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Dotted lines indicate the plasma membrane. (C) AtT20 cells were incubated for 3 min with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SST2R. Images were collected using standard confocal microscopy, and the area under the magnifying glass was acquired by STED microscopy. Scale bars represent 5 µm and 1 µm for the confocal and STED images, respectively. Dashed areas in the STED image indicate individual SSTR2-labeled vesicles. (D and E) AtT20 cells were incubated for 40 min with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SSTR2 (D) or syntaxin-6 (E). Images were collected using standard confocal microscopy and the area under the magnifying glass was acquired by STED microscopy. Scale bars represent 5 µm and 1 µm for the confocal and STED images, respectively. Dashed lines in the confocal image indicate the cell boundary and dashed lines in the STED image indicate tubulovesicular structures. (F) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp8]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification to the right for the [D-Trp8]-SOM treatment condition. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Images were acquired using an Abberior STED microscope as a single optical section through the cells and cropped using ImageJ. Dashed lines indicate cell boundaries. (G) AtT20 cells were fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification in the bottom row for each image. Scale bars represent 5 µm and 1 µm for the lower and higher magnification images, respectively. Images were acquired using an Abberior STED microscope as a single optical section through the cells and cropped using ImageJ. Dashed lines indicate cell boundaries. The arrowheads indicate the spatial segregation of immunoreactive signals of the indicated proteins.

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