Evaluation of lactic acid as a novel fixative for histological and neuroanatomical applications

Formaldehyde-based fixatives, particularly neutral-buffered formalin (NBF), are widely used in histology owing to their strong protein cross-linking capacity and reliable preservation of cytoarchitecture. However, formaldehyde is toxic and carcinogenic, motivating the search for safer alternatives. Here, we systematically evaluated lactic acid (LA) as a potential formaldehyde-free fixative for murine brain tissues. Brains were fixed either by immersion or by transcardial perfusion followed by immersion using LA at varying concentrations, pH-adjusted LA, or phosphate-buffered saline (PBS), and were compared with NBF. Histomorphological preservation was assessed using qualitative evaluation, semi-quantitative scoring, and quantitative crack-area measurements. Across all conditions, NBF provided the best overall tissue preservation. LA fixation showed clear concentration-dependent effects: low concentrations resulted in poor structural integrity, whereas higher concentrations (LA10-LA20) achieved moderate preservation of cytoarchitecture, reduced cracking, and acceptable cellular morphology. White-matter integrity remained inferior to NBF, consistent with the lipid-rich composition of myelin and the predominantly protein-denaturing, non-cross-linking fixation properties of LA. pH-adjustment experiments indicated that fixation efficacy partially depended on acidity. Notably, LA20 delivered by transcardial perfusion substantially improved tissue preservation compared with immersion alone and outperformed PBS. Although LA cannot fully replace NBF, high-concentration LA might represent an alternative for selected neurohistological applications.