# Antibody elution methods for multiplex immunofluorescence of Alzheimer's disease pathology in human post-mortem brain tissue

## TL;DR

This study compares antibody elution methods for staining Alzheimer's disease pathology in post-mortem brain tissue to improve imaging accuracy.

## Contribution

The study introduces a BME-based stripping buffer as a cost-effective alternative to TSA for multiplex immunofluorescence in brain tissue.

## Key findings

- BME-containing stripping buffer produced similar staining results to TSA-based methods in both short- and long-term fixed tissue.
- Traditional mIF remains limited by alignment risks during iterative imaging sessions compared to TSA.

## Abstract

Post-mortem human brain banks are a key resource for researching brain diseases. The New South Wales Brain Tissue Resource Center (BTRC) is a brain bank that focuses on neurodegenerative diseases, including alcohol use disorder and Alzheimer's disease. Most banks hemi-sect brains, freezing one half and fixing the other. Traditionally, formalin-fixed, paraffin-embedded tissue has been used for immunostaining, whereas frozen tissue has been used for complementary molecular studies. Immunofluorescent staining has been more difficult to employ than chromogen-based immunostaining in post-mortem brain tissue because of autofluorescence that is amplified further in archival tissue kept in formalin for long term storage. Multiplex immunofluorescence (mIF) is extremely useful for visualizing complex cell interactions in the brain but is limited by the availability of primary-secondary antibody combinations. Tyramide signal amplification (TSA) systems largely solved the latter issue but remains expensive to perform.

Given the increasing interest in human post-mortem brain tissue for mechanistic studies, we explored whether modifying stripping protocols for traditional mIF staining could improve performance to match newer TSA-based methods.

Employing β-mercaptoethanol (BME)-containing stripping buffer instead of heat-induced epitope retrieval gave similar results for both techniques in both short-term and long-term fixed tissue. However, iterative imaging sessions between cycles for traditional mIF still pose a greater risk for malalignment of target molecules in composite images.