In the previous units of this series, the immunohistochemical (IHC) methods of immunofluorescence and immunoperoxidase were described and discussed. These methods use antibodies to localize cellular proteins in tissue sections, which can then be visualized in either a dark field fluorescence microscope or routine bright field microscope. It is important to understand antibody structure and function in order to fully appreciate these immunohistochemical (IHC) techniques.
In the previous segment, immunofluorescence methods were discussed. These methods use antibodies labelled with fluorescein to directly localize cellular proteins in tissue sections, which can then be visualized in a dark field fluorescence microscope. Despite the advantages of this method, science is always in search of newer methods to gain more knowledge. Thus in 1970 Sternberger et al (1970) reported an improvement of Graham’s method (1965) using horse radish peroxidase enzyme (HRP) labelled antibodies to localize antigens and visualize them in the routine light microscope – bringing the method “to light” as one might describe. Now researchers and pathologists could see protein localization within the histology of formalin fixed paraffin embedded tissues viewed using a routine bright field microscope. This provided vast improvements over the immunofluorescence method.
Before we discuss immunofluorescence, we need to know what fluorescence is. There are certain substances, composed of molecules that will emit light when irradiated by a short wavelength, such as X-rays or ultraviolet (UV) light.
Anthony van Leeuwenhoek is credited with being the first person to use a microscope to view tiny “animacules” in 1674. At approximately the same time, Robert Hooke used a similar microscope to view thin slices of cork. The structures that he observed resembled the tiny cells that monks lived in at the local monastery, so he named the structures “cells”.
The most noticeable tissue processing artefacts are the wrinkles and tears in the tissue sections which are evident even at low power
Standard tissue processing may be carried out on any number of open and closed tissue processors, although closed processors are preferred due to safety concerns, both for the tissues and laboratory personnel.
The first four blogs of the troubleshooting series focused on being proactive with regard to the prevention of sub-optimal events in the histology laboratory
Some specimens may be very tiny; on the order of less than 0.1 cm. Some preparation methods employ the use of mesh cassettes, “tea bag” biopsy pouches, sponges, wrapping paper, etc. to contain the specimen and prevent it from escaping the tissue processing cassette.
No matter what type of histology laboratory you work in – hospital, research, reference, teaching facility – there will be times when you receive specimens that you do not normally receive.
In the previous blog we looked at one way to minimize troubleshooting by being proactive and looking ahead to possible situations and procedures that exist in your laboratory that may cause sub-optimal events. This blog will continue with that same mind set.