Week Four

This week, we will be testing out Barrow Neurological Institute's new cryostat for our immunohistochemistry procedure, as well as preparing the thermogradient for its new rat inhabitants.

In our experiment, we have multiple stages:

1. Habituation: We handle the rats (touch them, poke them, lift them) to get them used to human interaction and touch. We habituate the rats so that when we inject them with medication or we implant data loggers in them, they do not become agitated. 
2. Surgery: We implant data loggers into the rats' abdomens to accurately determine rats' core temperature. (See previous posts for more on data loggers)
3. Experimentation: We inject rats with medications that produce either an overwhelming hot, neutral, or cold temperature sensation. We observe amount and location of defecation, as well as frequency of shivering (in rats injected with medications that produces a "cold" feeling). This allows us to correlate our data. For example, a rat injected with "cold" medication should ideally seek the hot area of the thermogradient, as well as have increased defecation in the hot area of the thermogradient. We should also see shivering (a phenomenon that does not typically occur in rats) in the first few minutes after the "cold" medication is injected. See "Thermogradient Cleaning Procedure" post for cleaning procedure.
4. Surgery (Part 2): In this surgery, we selectively lesion or sham lesion the rat brain, with special attention on the BNST and POA.
5. Experimentation (Part 2): In this experiment, we follow the same procedure as in #3, however, we note the changes in rat behavior after lesions occur.
6. Surgery (Part 3): In this surgery, the rats are euthanized and their brains are preserved for study.
7. Immunohistochemistry: Here, we utilize the cryostat to slice each rat brain into tissue samples that can be mounted onto microscope slides.

To define immunohistochemistry (IHC), let us break down the word:

• immuno-: pertaining to production of antibodies or lymphocytes that can react with a specific antigen
• -histo-: relating to organic tissue
• -chemistry: study of matter

Therefore, immunohistochemistry refers to the process of detecting antigens in cells of a tissue section through a combination of anatomical, immunological and biochemical techniques. In other words, IHC is the process of identifying antigen-antibody interactions. In order to visualize an antigen-antibody interaction, we utilize IHC staining, or coloring of the tissues. 

We use IHC after using the cryostat (see "Week One" post) to slice cross sections of brain tissue. We will stain the tissue samples and use the stained tissues for data analysis.