Immunohistochemistry Protocols
IMMUNOHISTOCHEMISTRY PROTOCOLS
Your source for information, protocols, and techniques about immuohistochemsitry.
Immunohistochemistry is a molecular technique that combines principles from both immunology and biochemistry techniques and principles to the study of histology and pathology by revealing molecules and patterns within cells and tissues.
The first to describe immunohistochemistry was Dr. Coons. The original immunohistochemistry method consisted of an antibody tagged with a fluorescent probe which was developed in rabbits, which was mixed with tissue sections and searched for, under a fluorescent microscope following a period of incubation. Since, numerous advancements and improvements have been done, to make the technique of immunohistochemistry fairly inexpensive and indispensable in both pathology departments and molecular laboratory benches worldwide.
Numerous different procedures are available, however the most commonly used are the peroxidase-antiperoxidase immune complex method and more so, the biotin-avidin immunoenzymatic technique.
What Can Immunohistochemistry Do ?
Immunohistochemistry is a fabulous technique, which has numerous advantages and applications:
High sensitivity, high specificity, can be used on routinely processed tissue as well as with previously stained material, decalcified material can be used, all fixatives with the exception of a few can be used with immunohistochemistry, immunohistochemistry can be adapted to electron miscroscopy, andit can remove and replace the need for many stains.
Quick Labeling Method of Immunohistochemistry
The quick labeling method, which is approximately less than 10 minutes, was developed to assit pathologists in assessing sentinel lymph nodes during intraoperative assessment.
Sausage Tisse Block Antibody Testing Method
The sausage tissue block antibody testing method allows one to evaluate hundreds of tissue samples on a single slide with a drop of antibody. This method subsequently was used to form tissue microarrays. It was developed by Battifora.
Immunohistochemistry Protocol
Immunohistochemistry Protocol for Polyclonal Antibody
Primary Antibody
1. Take 4 micron paraffin sections down to water
2. Block endogenous peroxidase with 3% Hydrogen Peroxide for a total of 15 minutes incubation
3. Wash the sample in running water
4. HEIR Method
In a plastic coplin jar, immerse slides in 10mM of citrate buffer, pH of 6.0. The coplin jar should then be placed in a microwaveable pressure cooker surrounded by hot water. Microwave the sample for a total of 20 minutes.
5. Wash slides in water and then rinse in T.B.S. pH 7.6
6. Block with a concentrated blocking reagent for 10 minutes
7. Drain blocking reagent and apply antibody at optimized dilution concentration. Let incubate for 1 hour at room temperature
8. Wash with T.B.S. two times at 1 minute each
9. Drain T.B.S. and apply LINK at a dilution of 1:4 for 30 minutes at room temperature
10. Wash again with T.B.S. twice at 1 minute each
11. Drain T.B.S. and apply USA PO dilution 1:4 and incubate at 30 minutes at room temperature
12. Wash in T.B.S. twice for 1 minute each, then 1 at 5 minutes
13. Develop with a chromogen AEC for 10 minutes
14. Wash with water
15. Counter stain in Mayer's Haematoxylin
16. Cover sections with Crystal Mount
Basic Microscopy
Basic Microscopy
Setting the Ocular
Setting the ocular tubes is essential in order to best fit an individuals own inter-pupillary distance. You should focus on a section at the 10X power using only the right side ocular, by closing your left eye. Then you should repeat this with your opposite eye, using the left ocular, using the focussing device on the barrel of the left side ocular tube.This is done to suit the microscope for your own eyesight.
Setting the Iris Diaphragm
Setting the irish diaphragm is done in order to obtain the optimum illumination for your miscroscope. This is done be looking approximately 12 inches from the microscope into the barrel and closing the iris diaphragm of the condenser until the circle of light is cut down by one-third, leaving two-thirds of the illumination onto the objective lens.
Oil
Oil can be used to improve visibility when using the high power objective.
Histology Sections
A section in histology refers to an extremely thin slice of tissue. This slice is transparent because it has been shaved with a microtome (an instrument for slicing tissue into very thin slices e.g. 5 micrometres thin) from a tissue sample or gross specimen. The piece of tissue is then laid flat onto a glass slide, stained with a dye, which may or may not include antibody, and mounted with mounting fluid of the proper refractive index to allow for microsopic viewing of the specimen. Glass coverslips are added as a protective measure only. They do not provide a refractive index effective.
Method for the Preparation of a Histology Section
The mainstay of immunohistochemistry and pathological diagnosis is the use of the traditional light microscopy. In order to view tissue and individual cells, sections from tissue must be thin enough to allow light to pass through them and flow the image to the other optic. Unfortunately, the majority of tissues are far too soft and fragile to be cut thinly and therefore are immersed and absorbed with a more rigid and hardening solution which may support the matrix of the tissue, such as wax (paraffin) or plastic. These components are both repelled by water, and therefore water must be removed from tissue prior to supporting the matrix. Removal of water occures by gradual substitution with 100% alcohol. The alcohol is subsequently substituted also by toluene or xylol. These agents are soluble with both paraffin wax and plastic. The paraffin technique is the most widely used method of preparing tissue for histological examination with light microscopy.
The Paraffin Technique
The paraffin technique involves 5 steps: fixation, dehydration, clearing, infiltrating and embedding and sectioning.
Fixation
Fixation of tissue samples with formalin occurs almost immediately in order to prevent any deterioration and/or degradation of cells.
Dehydration
As mentioned previously, tissue samples are gradually passed through 100% alcohol in order to remove and replace all the water with alcohol.
Clearing
This step is required to replace the alcohol with xylol.
Infiltrating and Embedding
Paraffin wax is then used to replace the xylol.
Sectioning
This is the last step. The treated tissue sample is finally sections/cut with a microtome into thin, transparent sections that are between 5 to 10 micrometres thin.