A common Iba1 antibody labels vasopressin neurons in mice

Abstract

There are a wide variety of commercially available antibodies for labeling microglial cells based on different protein targets, as well as antibodies for the same protein target made in different species. While this array of targets and hosts allows for flexibility in immunohistochemical experiments, it is important to validate that different antibodies provide comparable and accurate immunodetection prior to experimental data collection. We found that a commercially available anti-Iba1 antibody, made in goat, produces irregular staining patterns in specific regions of the mouse brain, prompting a further investigation into the phenomenon. This Iba1-goat antibody displayed increased numbers of labeled cells when compared to expression of a CX3CR1-GFP reporter and IHC detection of P2RY12, two common microglial markers. Furthermore, immunodetection by other common anti-Iba1 antibodies made in rabbit and chicken did not display the excessive cell labeling when compared to the CX3CR1-GFP reporter. Upon further investigation, this Iba1-goat antibody was observed to highly colocalize with vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus of the hypothalamus (SON), the two main sites of vasopressin production in the brain. Other anti-Iba1 antibodies made in other species did not show this same colocalization with vasopressin. Finally, this effect was species-specific, as Wistar rats did not display erroneous cell labeling by the Iba1-goat antibody. In sum, the present study employs both qualitative and quantitative data to highlight the importance of validating antibody efficacy and specificity in a region- and species-specific manner. Significance StatementMicroglia are the primary immune cells of the brain and are involved in many neurodevelopmental, as well as neurodegenerative, processes, thus making the study of microglia an important area of neuroscience research. There is a wide array of antibodies available to label microglia. Specific detection of microglia using immunohistochemistry is crucial for understanding differences in cell density, morphology, and interactions with other cells in various contexts. In the present study, a common anti-Iba1 antibody made in goat was found to display erroneous labeling of vasopressin neurons in specific regions of the mouse brain, inconsistent with other microglial markers, which emphasizes the importance of validating antibody efficacy and specificity in a region- and species-specific manner prior to beginning experimental data collection.