Structural Biochemistry/Histology

What is it?

It is the study of tissues of plants and animals. Histologist usually use tissues, which are groups of specialized cells and promote staining onto these tissues after they are sectioned. There are typically two types of tissue section preparations. Usually one can freeze the tissue and then section them into 30-60 microns, or a “paraffin-embedded” method which is where hot wax is poured over the tissue of interest and molds into a block. The block is then sectioned using a microtome and the tissues are usually much thinner.

History

Marcello Malpighi a contemporary of Robert Hooke, scientist who discovered the cell was known to be the first histologist. He was able to use archaic microscopes from the 17th century and study the structure and component of tissues. However, another scientist named Marie Francois Bichat, who came after Malpighi introduced the word tissue and put the framework of many cells working collectively together for a specific function. Some consider Bichat to be the founder of present day Animal Histology. In modern day, histologist usually only collect four main types of tissues, which include the connective, epithelial, muscle and nerve tissues. It was not until after Bichat's death did the word histology began to circulte. Histology derives from two Greek words, Histo, meaning tissue, and logo meaning study. However, some consider Rudolph von Kolliker the actual father of Histology primiarly because of his work on creating an actual textbook and emphasizing tissues, this book is called "Handbuch der Gewebelehre".

Tissue fixation

Is the method by which scientist stop enzymatic activity and preserve the tissue in its most natural state. The fixatives, chemicals used in tissue fixation are usually toxic and will disinfect bacteria and parasites for a period of time. However, if upon fixating the tissue, overexposure to certain fixatives may lead to masking of proteins by masking epitopes. Usually, tissues are fixed by methods of immersion or perfused fixation. In order to do so formaldehyde is used in water. Paraformaldehyde is also available to be used as well.

Freezing

Is necessary in order to preserve the integrity of the tissue component and site of study. However, because much of tissue has water within in, another solvent must be used to drive water out as to not damage the tissue upon freezing. This can be done by using sucrose as solvent/media. Much like osmosis, as concentration of sucrose outside of the tissue increases water will rush out of the tissue to equilibrate the solution it is in. This process is called cryoprotection.

Sectioning slides

The method of sectioning varies from lab to lab, some labs use the paraffin technique while others use the freezing method.

Frozen Slide Method

Before samples are prepared they must be placed in sucrose for a relative day or two to drive the water out by osmosis. Upon dissection of the tissue of interest a section of the tissue must then be made and placed upon a container. Tissue freezing medium is then applied to the container and the sample is placed with it. Freezing under dry ice is then applied and then another layer of tissue freezing medium is placed so that it covers entire container. After letting the medium freeze for about half an hour, a square cut is made. The frozen cut is then placed on a mounting disc and tissue freezing medium is applied once again in order to mount the sample onto the disc. Once this is done and the disc is then mounted onto the microtome or cryostat sectioning can then begin. Upon mounting the disc on the cryostat, a blade is then applied to cut the sections. One must first however align the sample so that the cut is applied in a planar fashion. Cuts can be applied in various ways depending on how the user wants to obtain their sections.

References:

Tuszynski Lab - UCSD

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