Week 1: What do we know about immunohistochemistry and traumatic brain injury right now?

Shriya S -

Hello all once again! Before we delve into actual lab research, I want to first take some time in this post to discuss the current literature on traumatic brain injury (TBI) and immunohistochemistry (IHC).

The pathophysiology of TBI

Comprehending the pathology of TBI is the first step in understanding how enhancing the data analysis technique of IHC facilitates research on TBI pathology. The pathophysiology of TBI is incredibly complex. It includes neuronal membrane disruption, release of glutamate, affected glucose production, diffuse axonal injury, and neuroinflammation. Neuroinflammation, one of the most studied aspects of TBI pathology, involves the activation of glial cells, specifically astrocytes and microglia, which regulate functional recovery after TBI. It is important to note though, while inflammation may be protective acutely, in the long-term, it may exacerbate the injury. While research on the pathophysiology of TBI is robust, its pathology is very complex, highlighting the need for continued research.

Immunohistochemistry and TBI research

As mentioned, neuroinflammation has a prominent role in TBI’s acute and chronic pathophysiology. To study neuroinflammation, specifically cells such as microglia and astrocytes, researchers use IHC. IHC helps them understand how cells respond to impact acutely and chronically after injury; for instance, researchers found when healthy (ramified) microglia, which are very branched and have a small soma (cell body), get activated, they may take two forms: rod microglia–which are often found in the cortex and hippocampus, are less  branched, and have an elongated soma–and amoeboid microglia–which are found throughout the brain, are also less branched, and have a spherical soma.

Now that we know the role of IHC in TBI research and the basics of IHC (from the introduction post), let’s advance a little on our discussion of IHC. There are two types of IHC techniques: direct immunoassay, which involves only one primary antibody–the antibody that binds onto the antigen–and indirect immunoassay, which involves a secondary antibody binding onto the primary antibody. Direct and indirect immunoassays differ in their use of visualization methods: direct immunoassays typically utilize a substrate-enzyme binding process, such as horseradish peroxidase (HRP) with a substrate that emits visible light, while indirect immunoassays may employ fluorescence, where a fluorescent tag is attached to the secondary antibody to visualize the binding. The image below illustrates this.

There are two types of IHC techniques: direct immunoassay, which involves only one primary antibody–the antibody that binds onto the antigen–and indirect immunoassay, which involves a secondary antibody binding onto the primary antibody. Direct and indirect immunoassays differ in their use of visualization methods: direct immunoassays typically utilize a substrate-enzyme binding process, such as horseradish peroxidase (HRP) with a substrate that emits visible light, while indirect immunoassays may employ fluorescence, where a fluorescent tag is attached to the secondary antibody to visualize the binding.
Diagram that compares direct immunoassay and indirect immunoassay as types of IHC.

Optimizing the visualization of IHC tissue samples

Many factors are involved in optimizing the visualization of IHC tissue samples. These factors include antibody concentrations, incubation time, temperature, and antibody selection. This study will specifically analyze antibody concentrations, aiming to identify the optimal concentrations of primary and secondary antibodies used to investigate targets involved in post-traumatic brain injury (TBI) pathophysiology. Antibody concentrations are the focus, as opposed to other factors that influence the visualization of IHC tissue samples, because they are one of the most controllable steps of IHC.

Generally researchers test one dilution above and below the manufacturer-recommended dilution to determine the optimal antibody concentration. But, since this is not guaranteed to work, researchers are left to find optimal concentrations in literature (which is tedious and often futile), test on non-experimental tissue to determine the optimal concentration, or make do with sort of blurry tissue samples. Hence, by providing a reference of optimized antibody concentrations to future researchers, this study aims to facilitate TBI research and reduce animal use in the field.

This week: lab work

I will be going into the lab this Wednesday (2/12) and Thursday (2/13) to find the concentration of the IBA1 antibody, which stains for microglia (type of immune cell in the brain), that gives the most crisp and clear tissue sample. More information on my method of conducting IHC, analyzing tissue samples, and IBA1 results coming soon!

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Comments:

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    caitlin_e
    Hi Shriya! I am extremely intrigued by your project, and I am excited to learn more about your research methods later on. Are there any specific mathematical formulas you need to use to predict a possible optimal concentration of antibodies, or are your predictions based on other means?
    February 13, 2025 at 8:20 pm - Reply
    shriya_s
    Great question! I will be using a criteria to determine which tissue samples are considered visually optimized (their corresponding concentrations would then be the optimal concentrations). This criteria has qualitative (even concentration gradient, low background noise) and quantitative aspects (staining intensity, ratio of stained cells to total number of target cells). The quantitative parts don't require a mathematical formula, but rather use a scale. This criteria could change to only being qualitative for the sake of time, though. That is part of the actual method of my study. As for predictions, generally, I will be predicting the optimal concentrations of antibodies from other papers and general guidelines researchers use, instead of using mathematical formulas. Then, I will test them in the lab.
    February 14, 2025 at 9:59 am - Reply
    camille_bennett
    Hi Shriya, this is so interesting! Are there any limitation as to the severity of TBI you will study? Or are you looking at any type of TBI?
    February 18, 2025 at 12:54 pm - Reply
    shriya_s
    Hi Ms. Bennett, that's a great question! We are mainly investigating diffuse TBI. Diffuse TBI is a type of TBI that affects the whole brain, as opposed to a specific part of the brain (focal TBI). In terms of the severity, the brains of the rats we are studying have been subjected to around 320-340 millivolts of power, which I would classify as a moderate to severe intensity (based on what I've learned). Some brains have been exposed to less power, though, because of extraneous factors. Hope this answers your question!
    February 18, 2025 at 4:55 pm - Reply

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