Congratulations on purchasing a new autostainer! There are three parts to the start-up process: Instrument Verification, Stain Protocol Optimization, and Validation of the Staining Protocols. Instrument Verification – see CAP All Common Checklist (06/04/2020) COM.40350 Optimally, the company you purchase the instrument from should provide an operator manual; provide verbal procedural instructions; and test the […]
Stainers / Stains
The DNA of a nucleus directs the actions of the cell by instructing it to manufacture specific proteins. This “instruction” is sent from the nucleus into the cytoplasm by “messenger RNA” (mRNA).
In previous installments of the IHC Educational Series, we discussed the specifics of the basic procedures found in the IHC laboratory: immunofluorescence, immunohistochemistry and in situ hybridization. Even though these are different procedures, they all require the following support procedures.
The term “in situ” means “in the original place”, while “hybridization” refers to a hybrid composed of one strand of DNA with a complementary strand of nucleotides – which is referred to as a probe.
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”.
This blog is a followup to the previous article “The H&E Stain: Far From Routine”. In that article, the basics of the routine hematoxylin and eosin (H&E) stain were discussed. Now we shall discuss how to trouble shoot the routine H&E and how to ensure a high quality stain, once you have worked with your pathologist to determine the best stain result for your laboratory.
What exactly is a routine “H&E”? And what makes it routine? The first question is easy. “H” stands for ”hematoxylin” and “E” stands for “eosin”. Both are dyes used to stain tissue sections in histology. However, the procedure for correctly applying this combination of stains to tissue sections is far from routine.