# Longitudinal assessment of fluorescence stability shows fluorescence intensity decreases over time: implications for fluorescence microscopy studies

## TL;DR

This study shows that fluorescence intensity in IHC decreases over time, affecting data reliability and suggesting best practices for imaging.

## Contribution

The study reveals that fluorescence intensity decreases over time and is influenced by antibody selection and staining methods.

## Key findings

- Fluorescence intensity decreases over a six-week period in IHC.
- The extent of decrease depends on primary and secondary antibody choices and staining methods.
- These findings highlight the need for optimized imaging practices in fluorescence microscopy.

## Abstract

Immunohistochemistry (IHC) is one of the most widely used techniques across basic, translational, and clinical sciences. Key considerations need to be made to achieve reliable and robust IHC staining, however what has been understudied is the stability of IHC signal intensity over time. Changes in signal intensity over time have significant implications for data analysis and interpretation and ultimately impact scientific conclusions. In order to explore changes in IHC signal, the stability of fluorescence intensity was assessed over the course of six weeks using widefield or confocal microscopy. Results indicate that fluorescence intensity can decrease over this time course and that whether this decrease occurs and to what extent is influenced by the selection of the primary antibody as well as that of the secondary antibody, primary-secondary antibody combination, and utilization of chemical staining versus IHC staining. This investigation reinforces best practices for imaging fluorescent staining to ensure accurate and reliable data collection, be it for cell counting, assessing protein expression levels, or marker colocalization.