Abstract
Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome of the procedure. Here, we provide a detailed protocol suitable for thin cryostat sections, which has been successful for specific antibodies directed against key markers of hypothalamic neurons and glial cells. We include every detail concerning brain tissue collection, processing, sectioning, and labeling with optimal dilutions of antibodies with the aim of reducing non-specific background. Our background-optimized immunostaining protocol has been routinely used in the lab and allows efficient detection of specific neuropeptides, glial cells, and markers of inflammation and endoplasmic reticulum stress in the hypothalamus.