Week 2: Diving into the Deep End–Method, IBA1, and More
Shriya S -
Hey everyone! Welcome back to my blog. This week, I’m going to discuss my method, IBA1, and what I plan on doing in the upcoming week.
Method
Conducting immunohistochemistry, representative of science in general, takes some time. Usually, it’s a 2 day protocol. Here is a brief simplified summary of what we do on days 1 and 2:
Day 1
- Remove slide from freezer and rehydrate
- Antigen retrieval
- When we store the tissue, sometimes the antigens cross-link with the fixatives or get masked by them, so we have to go through steps that make sure the antigen can bind to the antibodies properly. This is known as antigen retrieval.
- Blocking
- This prevents nonspecific antibody binding, or the binding of the antibody to chemicals that are not the antigen.
- Add primary antibody
Day 2
- Add secondary antibody
- Block endogenous peroxidases
- Endogenous peroxidases are enzymes present in some cells that can lead to false-positive results.
- Add DAB solution
- This project uses a substrate-enzyme binding process to visualize the tissue (refer back to week 1’s post)–chromogenic staining; DAB, or 3,3′-diaminobenzidine, is the substrate/chromogen in this case. DAB is used, instead of fluorescence, because it lasts much longer.
- Dehydrate and coverslip
Now this protocol is just for conducting IHC on various tissue samples. As for analyzing it and determining which one is visually optimized, a criteria will be used:
Criteria for a visually optimized IHC staining:
- Minimal background staining.
- Combinative semiquantitative scoring is at least 9, and the individual numerical ranks for relative percentages of immunopositive cells and staining intensity are at least 5 and 2 respectively.
- Uniform concentration gradient through the entire sample.
Combinative semiquantitative scoring is calculated by considering both quantitative factors–the relative percentage of immunopositive cells in relation to the total number of target cells–and qualitative factors–the staining intensity. The relative percentages of immunopositive cells are assigned numerical ranks according to the following scale:
| Staining intensity | 0-9% | 10-19% | 20-29% | 30-39% | 40-49% | 50-59% | 60-69% | 70-79% | 80-89% | 90-100% |
| Score | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
Staining intensity will be scored from 0 to 3 (0, negative; 1+, weak positive; 2+, moderate positive; and 3+, strong positive). The final combinative semiquantitative score will be the sum of the numerical ranks of the relative percentage and the staining intensity. The numerical ranks for relative percentages of immunopositive cells and staining intensity must be at least 5 and 2, respectively, to prevent discrepant scoring–e.g., when the relative percentage of immunopositive cells is low but the staining intensity is high enough so that the sum of the scores is at least 9–between the two parameters.
Whichever tissue sample meets the criteria most completely would be the visually optimized stain, and the antibody concentration(s) that resulted in that stain would be considered the ideal concentration for that cell type/tissue.
Now, this is the criteria as of now, but as I was discussing with my mentor last week, quantifying some aspects of the criteria may be difficult in terms of time. To determine the ratio of stained cells to the total number of target cells, for instance, may require more time than we have. So, this criteria is definitely subject to change, but the concepts underlying the criteria should remain the same.
IBA1
Last Wednesday and Thursday, I was in the lab, and I got to meet the amazing team and experience an IHC protocol being conducted in front of me! IBA1 (ionized calcium binding adaptor molecule 1) is an antigen found on microglia (a type of glial cell with neuroinflammatory functions in the brain). Last week, we used antibodies that bind to this antigen to stain for microglia in various tissue samples. From previous research (as I discussed in the last post), we know that microglia can change their morphology (shape) when they get activated as a result of traumatic force. IHC has shown us they can take on a rod-like shape or an amoeboid shape, both of which we do not comprehend completely (hence, why we research). Once we analyze the stained IBA1 slides this week, I can show how a rod-shaped microglia would look compared to a amoeboid or ramified (resting/healthy) microglia!
This week: lab work
Last week was such an exciting experience! I don’t think I’ve been around equipment that expensive before (one primary antibody flask with a couple hundred of microliters can cost hundreds of dollars!). This week, along with analyzing the IBA1 tissue samples from last week, we will be conducting IHC with antibodies that target the antigen GFAP (glial fibrillary acidic protein). GFAP is mainly found on astrocytes (another type of glial cell much bigger than microglia and involved in neuroinflammation). But more information on that in the next post.
I will also have the opportunity to witness rat and mice surgeries (i.e., researchers inflicting TBI onto rodents in a controlled way for research) this week. I think instead of avoiding the fact that this is research being done on animals, it’s important to understand and know all that is happening–the good and the ugly–to respect their lives and maintain empathy amidst research.
I’m very excited for this week! Stay tuned for more information on GFAP, rat/mice surgeries, and IBA1 stain results!
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