Figure 8. Assessing movement of the GPIHBP1-specific antibody 11A12 from the abluminal plasma membrane (APM) to the luminal plasma membrane (LPM) in brown adipose tissue (BAT) capillary endothelial cells of living mice.

Alexa Fluor 488–11A12 (green) was injected into the interscapular BAT of Gpihbp1^+/+ and Gpihbp1^S/S mice. After 15 or 90 minutes, images of capillary cross sections containing an endothelial cell nucleus (blue) were recorded by fluorescence microscopy. The presence of the cell nucleus made it possible to visualize 11A12 on the APM (blue arrowhead) and the LPM (magenta arrowhead). Shown here is a representative capillary cross section for each experimental condition. On the right, we show the number of capillary cross sections (from a total of 50 counted) in which Alexa Fluor 488–11A12 was detectable at the capillary lumen. Three additional cross sections per experimental condition are shown in Supplemental Figure 9. (A and B) Capillary cross sections in BAT from Gpihbp1^+/+ and Gpihbp1^S/S mice 15 minutes (A) or 90 minutes (B) after the injection of Alexa Fluor 488–11A12. (C) Capillary cross sections in BAT 90 minutes after an injection of Alexa Fluor 488–11A12, 0.75 U heparin, and 15 μg dextran sulfate. (D and E) Capillary cross sections in BAT 90 minutes after an injection of Alexa Fluor 488–11A12 and 34 μmol of a synthetic peptide corresponding to the WT GPIHBP1 AD (EDGDADPEPENYNYDDDDDEEEEEE) (D) or the S-protein tag (KETAAAKFERQHMDS) (E). Scale bars: 2 μm.