The end goal of the initial histology lab today is to begin

The end goal of the initial histology lab today is to begin to recognize organ systems at the tissue level by sight under the microscope. This will be eventually accomplished by you next lab using hematoxylin & eosin staining, and by trichrome staining. Stop and Think: What makes a good image in which two or more features of that image are in sharp relief? Why? How must a chunk of tissue (ex: biopsy) be obtained and processed? How is that chunk further processed before it can be stained as a thin tissue section on a glass slide? What are the major steps in preparing a thin tissue section for staining, regardless of specific method?

Solution

In counter staining and differential staining techniques, we aim to stain areas of interest with a stain which contrasts with the backgroud stain. All the stains used must be used adequately, i.e., no parts of the stained tissue must be over-stained or under-stained, so that the different features can be observed. The fixing of the tissue to the slide must be done properly so that the tissue does not displace during staining, transport or storage. The tissue should be free of water, and all equipment used such as the slide, cover slip and the lenses much be clean and free of blemishes to prevent distortion of the image.

For obtaining the tissue for staining, an incisional biopsy is preferred. This type of biopsy retains the histological structure of the tissue, helping us to recgnize various cellular patterns in the tissue. The tissue is excised from the organism and is first fixed to prevent decay and changes in structure. Fixation can either be chemical or physical (by freezing). Chemical fixation is achieved in the most cases by using formalin, which is solution containing approximately 4% formaldehyde in a neutral buffered saline. It works by creating cross-links in the proteins in the tissue, primarily between lysine residues. Glutaraldehyde works similarly but can link more distant proteins, while not being able to penetrate deeper in to the tissue due to its size. A combination of both may also be used. Other techniques for chemical fixation include protein precipitaion, oxidation by osmium tetroxide, Hepes-glutamic acid solvent etc. Fixation by freezing is achieved by treatment by a a cryoprotective medium such as OCT, TBS, glyceral or DMSO, and rapid freezing using liquid nitrogen. It is then fixed in either acetone or ethanol.

Before being transferred onto a slide, a number of steps must be followed. Processing of the sample is done to solidify and remove all the moisture from the sample. This is done by using paraffin wax or resins and progressive baths of alcohol. The processed sample is then embedded agents such as agar, gelatine or wax, and hardened. This makes the sample easy to section and improves shelf life. Sectioning of the sample is done to prepare slices of the sample for crossectional viewing. 4 millimeter sections are cut using a steel knife for mounting on a glass slide for light microscope.