Journal: Journal of Biological Chemistry

Article Title: Effect of Human Carbonic Anhydrase II on the Activity of the Human Electrogenic Na/HCO3 Cotransporter NBCe1-A in Xenopus Oocytes

doi: 10.1074/jbc.m602181200

Figure Lengend Snippet: FIGURE 6. Immunolocalization of EGFP and CA II domains in oocytes expressing EGFP-e1-CAII or EGFP- e1. Three days after injection of cRNA encoding EGFP-e1-CAII or EGFP-e1, oocytes were prepared for immu- nocytochemistryasindicatedunder“ExperimentalProcedures.”Preparationswereincubatedovernightsimul- taneously with mouse monoclonal anti-EGFP and with rabbit polyclonal anti-human CA II. The secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 594-conjugated goat anti-rab- bit. A-C, Alexa Fluor 488-labeled images were taken with excitation/emission filters set at band pass 488 nm/long pass 505 nm to detect EGFP signals. D-F, Alexa Fluor 594-labeled images were taken with excitation/ emission filters set at band pass 543 nm/long pass 560 nm to detect CA II signals. These images show that EGFP-e1-CAIIandEGFP-e1arecorrectlytargetedtotheplasmamembrane.G-I,mergesoftheimagesinthefirst and second columns. G shows that the EGFP staining (green) of EGFP-e1-CAII overlapped with the CA II staining (red). J–L, Nomarski images taken simultaneously with either the EGFP or CA II images. Bar 50 m.

Article Snippet: Slides were blocked by washing (once for 15 min) in TBS 0.1% BSA 10% normal goat serum (Vector Laboratories, Burlingame, CA).We incubated the preparations with mouse monoclonal anti-EGFP (BD Biosciences; 1:100 in TBS 0.1% BSA 10% goat serum) and rabbit polyclonal anti-human CA II (Rockland, Gilbertville, PA; 1:1000 in TBS 0.1% BSA 10% goat serum) overnight.

Techniques: Expressing, Injection, Labeling, Staining