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Techniques in immunocytochemistry

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Techniques in Immunocytochemistry
ABSTRACT
According to Burry (2010), Immunocytochemistry is a useful method to identify antigens and cells. Two tissue sections are required to show that label localization is correct. The primary antibody shows the specificity of binding to the antigen; the secondary antibody shows the label is specific to the primary antibody. Immunoglobulin G (Ig G) is the most common antibody type used in immunocytochemistry. The variable region (Fab portion) of the antibody binds it to the epitope part of the antigen. In this practical, the tissue sections were blocked and incubated, and also two plastic petri-dishes were labelled, one as a control and the other an antibody. After that both slides were incubated in a medium containing diaminobenzidine (DAB). The sections were mounted with a minimal volume of aqua mount and were then visualized under a light microscope. The tissue that was incubated with primary antibody was given a blue nucleus with a brown coloured background; this indicates the presence of the target antigen (Griffins, 2011).
INRTODUCTION
Immunocytochemistry is a common laboratory technique that makes use of antibodies to target specific antigens in a cell via specific epitopes (an epitope is the part of an antigen that is recognized by antibodies). These bound antibodies can then be detected by using many different methods. The antigen is bound by a primary antibody, which is then amplified by use of a secondary antibody. The secondary antibody can be an enzyme that is conjugated. Immunocytochemistry binds antibodies that are specific reagents and allow unique detection of proteins and molecules. It is a valuable tool for the determination of cellular content from individual cells (Gillian et al., 2011).
In this practical, a specific monoclonal antibody was used to detect a tissue antigen. An immunoperoxidase staining was used to view the location an antigen within the tissue.
METHOD
The tissue sections of the cardiac myosin were cut and fixed in acetone. After this the slides were unwrapped and each slide was placed in a plastic petri-dish. The two plastic petri-dishes were labelled; one was the control and the other the antibody. A piece of paper was put underneath each slide to form a humidity chamber. The tissue sections were blocked by adding two drops.
A PBS buffer containing three percent BSA was introduced and then the tissue incubated at room temperature for five minutes. After that the excess medium was tipped and the zone around the tissue section was wiped dry with paper towelling. The culture supernatant of 50 µL was added to the antibody slide, while 50 µL PBS containing three percent BSA was added to the control slide; both slides were incubated at room temperature for a total of 40 minutes. Later both slides were tipped of excess medium and then washed by immersing sections through a PBS buffer in a coplin jar. Both slides were washed for two minutes, each in a fresh medium, and then the zone around the tissue section was carefully wiped dry.
Added to both slides was 50 l of HRP of the conjugated secondary antibody. The slides were then incubated in the moist chamber at room temperature for 30 minutes. After that both slides were tipped of any excess medium and then were washed by immersing each section in a PBS buffer in a coplin jar. Both slides were washed for two minutes each in a fresh medium, and then the zone around the tissue section was carefully wiped dry.
After that both slides were incubated for up to six to eight minutes in a medium containing diaminobenzidine (DAB) and then placed in a petri-dish. Both slides were rinsed in tap water to remove any excess chromogen, counterstained with a drop of haematoxylin each for between 20 and 60 seconds, and rinsed with distilled water. Both slides were visualized under magnification with A 40 light microscope. After a recording of what had been seen, then the sections were mounted.
RESULT
In this practical the antibody targeted was cardiac myosin; the primary antibody control was a specificity control and confirms that the primary antibody binds to the correct antigen on the tissue or cells. The secondary antibody control shows that the labelling observed is due to binding of the secondary antibody to the primary antibody. In this practical, the tissue that was incubated with the primary antibody was given a brown background colour, whereas the control tissue was given a blue background colour.
Figure 3: Schematic Diagram of immunochemistry
Figure 1: Control tissue section under a light microscope (negative result)
Figure 2: Primary tissue section under a light microscope (positive result)
DISCUSSION
In the antibody slide, when we visualized the tissue section under the light microscope magnification of A40 we were able to see a dark-brown colour because of the medium containing diaminobenzidine (DAB). The section tissue, which was targeted by a primary antibody and then a secondary antibody (HRP conjugated), was conjugated with a peroxidase enzyme. This bound DAB as a substrate and oxidized it by producing an observable brown colour. The observable brown colour indicated the presence of the targeted antigen. For the control slide we were unable to observe the brown colour, which indicates the absence of the targeted antigens. For the control slide we added 50 µl of phosphate buffered saline, which washed the solution and blocked the surface to prevent non-specific binding to the myosin protein from being approved.
CONCLUSION
This practical experiment showed positive results, as the antibody slide was visualized under a light microscope producing an observable brown colour. This demonstrates that secondary antibody binds to the primary antibody and reveals the location of the antigen.
Crocker (1994) suggests that most antibodies suffer from some limitations, such as that the molecules which they detect are only associated with T or B cells.
REFERENCES:
Burry, R. (2010) Immunocytochemistry. A practical Guide for Biomedical Research. New York. Springer.
Crocker, J. (1994) Molecular Biology in Histology. West Sussex. Chischester.
Gillian, R, B. and Petrus (2011) Techniques in Immunocytochemistry. USA, Califorrnia. Academic Press.
Griffins, G. (2011) Fine Structure Immunochemistry. London. Limited.

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