# Immunohistochemical Detection of Microglia Using Iba-1 as a Marker in Health and Disease

## TL;DR

This study shows that Iba-1 is a reliable marker for identifying microglia in both healthy and diseased brains, though it has some limitations in distinguishing microglia from other cells.

## Contribution

The study demonstrates the utility and limitations of Iba-1 as a marker for microglia in various species and conditions.

## Key findings

- Iba-1 is present in microglial cells across different brain regions and developmental stages in rats and humans.
- Iba-1 staining reveals structural and functional differences in microglia under normal and pathological conditions.
- Iba-1 cannot distinguish microglia from infiltrating macrophages, suggesting the need for multi-marker approaches.

## Abstract

The aim of the study was to evaluate the possibilities and limitations of using the immunohistochemical detection of Iba-1 protein for morphofunctional analysis of microglia under different conditions.

In the study we used brain samples from Wistar rats at different ages: 7 days of postnatal development (n=18), 14 days of postnatal development (n=18), 4–6 months (n=22); the brain samples from 3–6-month-old SHR (spontaneously hypertensive rats) male rats (n=4); human cerebral cortex samples (n=10). Rabbit polyclonal antibodies against calcium-binding protein Iba-1 (Biocare Medical, USA) were used to detect microglia immunohistochemically followed by light and confocal laser microscopy. A previously developed original technique was applied to simultaneously detect microglial cells and amyloid plaques.

Iba-1 protein in microglial cells was shown to be present in grey and white matter in all cerebral regions under study. Microglial cells in different regions were noted to be characterized by pronounced structural and functional features when stained for Iba-1. In addition to microglial cells, in the rat brain there were found other Iba-1-immunopositive macrophages, which were tissue cerebral macrophages differing from microgliocytes by specific morphology and localization. Iba-1 protein was demonstrated to be present in microglial cells in all investigated stages of postnatal development that makes Iba-1 an appropriate marker for comparative ontogenetical studies of microglial cells. The localization of Iba-1 in the bodies and processes of microgliocytes enables to more clearly identify the complex ramified cell morphology and make three-dimensional reconstructions. In the brain of spontaneously hypertensive rats (SHR line rats) the Iba-1-immunopositive microglia were shown to have a number of structural and functional features indicating the moderate activation of microglia. 48 h after ischemic injury, an anomalously great number of large Iba-1-immunopositive cells with amoeboid morphology were detected in the gray matter of the striatum in an ipsilateral hemisphere near the injured area in the rat brain. Microglial cells in human cerebral cortex were noted to be localized in the majority of the detected amyloid plaques and characterized by amoeboid morphology indicating their severe activation.

The presented findings suggest Iba-1 protein to be a reliable and universal microglial marker. The immunohistochemical detection of the protein enables to identify and analyze qualitatively and quantitatively microglial cells in different brain regions in human and laboratory animals under normal and pathological conditions. The limitations of using Iba-1 as a marker of microglia include the inability to determine the microglial activation vector and the difficulty in distinguishing between microglia and infiltrating macrophages of the brain in pathology. In such cases, it is necessary to improve the technology for detecting microglia that can be based on using a multi-marker analysis.