Pepsin pretreatment allows collagen IV immunostaining of blood vessels in adult mouse brain

While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods could improve vascular staining in the adult mouse brain. We found that pepsin digestion prior to immunostaining unmasked widespread collagen IV staining of the cerebrovasculature in the adult mouse brain. Pepsin treatment also unmasked widespread vascular staining with laminin, but only marginally improved isolectin B4 staining and did not enhance vascular staining with fibronectin, perlecan or CD146. Collagen IV immunoperoxidase staining was easily combined with cresyl violet counterstaining making it suitable for stereological analyses of both vascular and neuronal parameters in the same tissue section. This method should be widely applicable for labeling the brain vasculature of the mouse in aldehyde fixed tissue from both normal and pathological states.