Effect of Immunohistochemistry on Molecular Analysis of Tissue Samples

TLDR

Laser‑based tissue microdissection is a critical tool for molecular analysis of histological sections, and recent antibody‑guided, automated approaches have improved its precision while raising questions about how staining affects biomolecules. The authors sought to determine how immunostaining influences DNA, RNA, and protein integrity in microdissected samples. They performed DNA, RNA, and protein analyses on immunostained, microdissected tissues. DNA remained largely intact but yielded 50–75 % less material, RNA was degraded by endogenous RNases, proteins were amenable to electrophoresis and mass spectrometry but less so to solution assays, indicating that immunoguided microdissection is viable but requires protocol refinement.

Abstract

Laser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the 1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno–laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods.