Histology Lecture #1 January 13, 2014 Tissue Preparation Most of the tissue that we will be observing was prepared

using these techniques. 1. Acquire the tissue 2. Immediately fix the tissue placed in a solution that will cross link the molecules that comprise the cellular structure. It preserves the specimen in its natural form. It is extremely important! a. Formaldehyde is a common fixative agent 3. Dehydration accomplished by passing the fixed specimen through increasing concentrations of alcohol (ethanol). Start at a low percentage (about 75%) and progress to 100% 4. Clear the alcohol (removal of alcohol) Toluene or Xylene will void the alcohol from the sample. 5. Before embedding, you must infiltrate the sample with paraffin or some type of wax. You take a sample and place it in a mold containing paraffin and you increase the temperature. Incubation allows the paraffin to infiltrate the tissue. 6. You remove it from the mold and allow it to cool (allowed to harden). This is called embedding. 7. You trim the paraffin away from the tissue, making it as small as possible. So you have this hard piece of material with the tissue embedded. 8. Using a microtome to make extremely thin slices of the sample. With a microtome, you can make cuts as thin as 1 m in thickness. This allows you to make slices between 1 and 10 m. These thicknesses are acceptable for light microscopy. If you are preparing a sample for electron microscopy, instead of using a microtome you would use an ultra-microtome, which allows for even thinner (<1 m). Likewise, paraffin wax is not used for electron microscopy. Instead, an epoxy resin is used. Epoxy resin is much harder and allows for thinner slices to be produced. Our slides are already prepared! So we wont do any of this stuff. However, during this process, quite a lot of things can go wrong these are called artifacts. These will be discussed later. We must be able to identify artifacts! Not being able to identify artifacts can lead to misdiagnoses. It is important to develop these skills (separating tissue from artifacts). Although most of our commercially prepared slides are free of artifacts. Once the tissue is prepared (steps above), you take the sample and you float the sample in a water bath to open it up and once opened you spread it onto a slide. And before staining this is what your slide would look like (find a picture) Very difficult to distinguish structures! [The image that is shown is of neural crest cells] These are multipotent embryonic cells. The first panel shows you the unstained light micrograph. The second panel shows a phase-contrast micrograph (you can see some membranes and nuclei some structures). The third panel shows differential interference contrast microscopy (similar to phase-contrast). This type allows you to see a raised image (somewhat 3-D). Take home message: staining makes things easier to see!

[Another example of unstained vs stained] Gingival Epithelium (gum epithelium). This sample is stained with methylene blue. Methylene blue is a basic dye. Dyes used in histology are either acidic compounds or basic compounds. Basic compounds are attracted to components in the tissue/cells that are negatively charged. DNA is negatively charged. Therefore, methylene blue (being a basic dye) will be attracted to DNA. Hence why in this example, the nuclei are stained blue (you can also see some bacteria stained too). Components that bind to basic dyes are said to be basophilic Components that bind to acidic dyes are said to be acidophilic o Proteins are positively charged. o Proteins are said to be acidophilic because they bind to acidic dyes.

The most common type of staining used in histology is hematoxylin-eosin (H&E) stain. This is a combination of two dyes. Most of the figures in our textbook and atlas utilize this dye. This contains both acidic and basic dyes: Hemotoxylin is basic o Stains the nucleus and DNA a dark purple or blue color Eosin is acidic o Stains the cytoplasm pink

The slide above is of kidney cortex and you can see the glomerulus and Bowmans capsule (within the renal corpuscle). The cytoplasm is stained pink. Eosin will also stain collagen fibers and muscle fibers pink. Depending on what tissue you have isolated, histologists must choose an appropriate stain depending on what structures they are trying to identify. Also, if you are trying to see as much structure as possible, it is important to do different types of stains and different samples. [Here is another example of an H&E stained sample] This is cardiac muscle. How do we know? First, we can see the striations (which is also common in skeletal muscle). Intercalated discs are also seen! Again, the nuclei are stained dark purple.

[This is a slide of lung tissue: late stage and early stage emphysema] Here you can see the effects of H&E staining on a sample. The purple nuclei and the pink ECM. You can also see a blood vessel here (it was identified as a vein due to the cell wall being very thin no smooth muscle).