Simultaneous Immunofluorescence-Based In Situ mRNA Expression and Protein Detection in Bone Marrow Biopsy Samples

Fluorescence in situ hybridization (FISH) can be employed to study the expression and subcellular localization of nucleic acids by using labeled antisense strands that hybridize with the target RNA or DNA molecules. Likewise, immunofluorescence antibody staining (IF) takes advantage of the specific interaction between a fluorophore-labeled antibody and its corresponding antigen. This protocol reports the combination of RNA-FISH and IF antibody staining for simultaneous detection of both RNA transcripts and proteins of interest in routine formalin-fixed paraffin-embedded (FFPE) bone marrow biopsy samples. Herein, we provide a detailed description of the methodology that we have developed and optimized to study the spatial expression of two transcripts-TGFB1 and PDGFA1-in human hematopoietic (CD45⁺) and non-hematopoietic (CD271⁺) cells in the bone marrow of patients with acute lymphoblastic leukemia (ALL). Key features • The protocol describes the simultaneous visualization of RNA target transcripts and protein expression. • In situ RNA and surface marker analysis was established for routine human bone marrow biopsies.