Immunohistochemistry (IHC) is a standard method for spatial proteomics and allows for exploration of protein expression at single-cell resolution within the intact tissue environment. Stringent procedures and proper antibody validation strategies are however needed to ensure reliability of results. Application-specific strategies have been proposed by the scientific community to ensure high quality despite variations in sample preparation between different antibody-based methods. Here, the entire workflow utilized within the Human Protein Atlas, from sample preparation to annotation of the IHC staining patterns is described in detail, with important notes on various factors that can affect the outcome of IHC. Methods include tissue microarray (TMA) production, tissue sectioning, IHC, annotation, and validation. Also, building on previously suggested validation strategies, IHC-specific orthogonal and independent validation methods are outlined.