Sectioning
A microtome was used to section the canine brain. At the manual operation of the scientist, this machine uses an extremely sharp blade to cut slivers that are 10 microns thick from a block of brain that was previously mounted in paraffin wax. The blade is so sharp that an additional clearance for handling sharps was required in order for me to use the microtome.
Once the section of tissue was cut, it was set in a hot water bath to flatten and then it was eventually collected on a slide. Typically, two small sections fit on one slide. The slides were then set out to dry and then baked in an oven overnight at 40℃ in preparation for the staining step the following day.
Although sectioning was quite meticulous and patience was certainly necessary, I was able to get the technique down and successfully cut and create over 150 slides.
Histochemical Staining
Once on slides, the tissue was stained in order to detect and locate iron, which is associated with the neurodegeneration that causes dementia and Alzheimer’s disease. We used a histochemical staining technique that has been used since the 1800’s in order to stain for iron in brain tissue. This stain uses a chemical called potassium ferrocyanide that reacts with iron to form a blue crystal called Prussian Blue.
We used a modern version of this technique that is enhanced by DAB (3,3’-diaminobenzidine), which reacts with hydrogen peroxide in the presence of Prussian Blue and turns the surrounding tissue brown. The brown stained tissue thus indicates accumulated iron in the CCD brain.
I focused on iron staining and successfully stained all of the sections that I cut (and some sections that AJ had previously cut). While I honed this technique, other various pathologies in the dog brain were stained as part of the larger project. The other pathways explored include tauopathy and amyloid-beta, other common neurodegeneration markers.
After the iron stain, the slides were stained with hematoxylin which acted as a counterstain, turning the rest of the tissue blue to give context to the iron stain. The difference in color is what would be eventually used quantify data.
Imaging Slides
After being stained, mounted, and set to dry, the slides were imaged with an Olympus VS-120 Slide Scanner. This machine uses an automatic mechanical claw to grab and place slides on the scanner in order to image up to 100 slides at a time, producing extremely high definition images of the tissue.
We faced some technical difficulties in June when the slide scanner kept “dropping” slides. This was very annoying because it could have only been two slides in when the error popped up and ceased further scanning. If it was meant to run over the weekend in order to scan all 100 slides, we found that we were 98 slides behind when we came in on Monday.
Overall, the issue wasn’t very severe and microscope maintenance was able to fix the scanner in order for it to run smoothly and efficiently again. After a couple days of intense slide imaging, we were back on track with his timeline.
Although, I was not formally trained to operate the Olympus VS-120 Slide Scanner, I observed someone else operate it and I helped load the slide trays.
Tissue Analysis
Once the slides were imaged and in a high definition photo format, ImageJ software was used to analyze them. To quantify our results, we created an algorithm. Using color deconvolution, we separated the blue and brown in the tissue and created an image of only the brown (iron staining) from the existing image. From there, a threshold of different levels of intensities were created to separate the different kinds of brown staining. High brown intensity correlated with cellular and vascular deposits, while low brown intensity correlated with diffuse white matter staining.
We calculated a total tissue area by adding together high brown and blue staining. With this, the percent area of high and low intensity staining was calculated.
Other Useful Skills
Besides the techniques I was formally taught, I was able to practice and master many other lab skills. In my labs at Washington & Jefferson, I was introduced to new materials such as pipettes, microscopes, and dangerous chemicals and new techniques like working in a fume hood and calculating dilutions. Even though these materials and techniques are simple and I had no issue with them at W&J, I am now far more comfortable with them. Overall, I am drastically more confident and efficient in the lab than I was when I first started my internship.
I was taught to use new equipment as well. I now know how to use a pipette gun and a pH scale and how to mount slides. The protocol I followed was very specific and required attention to every small detail, so I got lots of practice handling chemicals and measuring exact amounts. Everything I learned during my internship is useful for future lab experiences.