Immunofluorescence Detection of Lysyl Oxidase in Formalin-Fixed Paraffin-Embedded Breast Tissue.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that catalyzes the oxidative crosslinking of collagen and elastin in the extracellular matrix, contributing to tissue stiffening and remodeling in the tumor microenvironment. Dysregulated LOX expression has been mechanistically linked to breast cancer invasion, pre-metastatic niche formation, and poor clinical prognosis. Here we describe a complete protocol for evaluating LOX protein expression in formalin-fixed paraffin-embedded (FFPE) human breast tissue sections, comprising (1) hematoxylin and eosin (H&E) staining for morphological assessment and tissue region selection and (2) direct immunofluorescence (IF) detection of LOX using an Alexa Fluor 647-conjugated anti-LOX rabbit recombinant monoclonal antibody (clone EPR4025), which eliminates the need for a secondary antibody step, with antigen retrieval performed under pH 6 citrate buffer conditions. The protocol is validated on ductal carcinoma in situ (DCIS) and DCIS-adjacent breast tissue and is readily adaptable to other FFPE tissue types and directly conjugated fluorescent antibodies.