Biotin-containing proteins of the insect nervous system, a potential source of interference with immunocytochemical localization procedures

Abstract When the biotinylated Manduca sexta adipokinetic hormone gene was used as a probe for in situ hybridization, the intrinsic neurosecretory cells were stained with a biotin detection system that contained streptavidin or avidin. Further experiments showed that the DNA probe was not necessary for staining these cells by streptavidin-alkaline phosphatase, and that they were not stained by alkaline phosphatase alone. Similarly, the intrinsic neurosecretory cells were stained directly by streptavidin conjugated to a fluorescent dye. Other parts of the central nervous system could also be stained with streptavidin-alkaline phosphatase but not as readily as the intrinsic neurosecretory cells of the corpora cardiaca. Further analysis demonstrated three biotin-containing proteins in the intrinsic neurosecretory cells of the corpora cardiaca and in the brain. The most abundant of these proteins, when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was found to have a molecular weight of 130,000, which is the size of the subunits of pyruvate carboxylase, a biotin-containing enzyme. The same protein was recognized by an antiserum against an insect pyruvate carboxylase, indicating that this protein is probably pyruvate carboxylase. The results reported here indicate that the intrinsic neurosecretory cells of the corpora cardiaca may contain pyruvate carboxylase in a concentration higher that other cells of the central nervous system. We also note that caution is necessary to avoid false positive results if an avidin containing detection system is used for in situ hybridization or immunocytochemistry.