313 - University of Western Australia
3313. Immunohistochemistry Laboratory Practical.
Thea Shavlakadze & Anil Sharma
Outline:
1) An unconjugated primary antibody to smooth muscle (-actin binds to the antigen in smooth muscle cells and pericytes.
2) The primary antibody is labelled with a biotinylated secondary antibody.
3) The bound biotin molecules are detected with enzyme-labelled streptavidin (alkaline phosphatase conjugated).
4) The chromogen-substrate solution (BCIP/NBT substrate) then reveals the alkaline phosphatase molecules bound to streptavidin.
Details:
1) The primary antibody (monoclonal mouse anti human smooth muscle (-actin) was raised in mice against human smooth muscle (-actin. Smooth muscle (-actin is a cytoskeletal protein found in pericytes and smooth muscle cells. Therefore, this protein can be localized in tissues containing abundant smooth muscle cells (e.g. gut and large blood vessels), and pericytes (capillaries and venules).
2) In the second step, anti-mouse/anti-rat immunoglobulins conjugated to biotin are used to detect the primary antibody. Biotinylation is a mild process in which biotin is covalently linked to the secondary antibody.
3) The third step uses the strong affinity of streptavidin for the biotin molecule. A solution containing enzyme-labelled streptavidin is applied, and the streptavidin binds strongly to the biotin.
4) The colour reaction is developed using a substrate-chromogen solution, resulting in a coloured (dark blue) precipitate at the antigen ((-actin) site.
Method:
(Please work in groups of 2 or 3).
You are provided with one slide on which there are two tissue sections. The top section will be labelled with the primary antibody; while the bottom section will be a negative control. The tissues were immerse-fixed in 10% phosphate-buffered formalin, then processed and embedded in paraffin wax. Sections were cut with a microtome at 5µm in thickness. Sections have already been dewaxed and rehydrated (xylene – alcohol – water).
1) Tap off any excess water and carefully draw a wax circle around each tissue section.
2) Apply primary antibody to the top tissue section. The primary antibody is diluted 1:50 in PBS+ 1% BSA+ 0.1% Sodium azide. Apply (approx 50µl) so that the top section is covered. Apply dilutant (no primary) to the bottom section; ensure that the two solutions do not mix. Incubate for 10 minutes.
3) Wash well in PBS (3x 5minutes).
4) Apply biotinylated link antibody (yellow solution) to both sections. This solution does not need to be diluted. Apply (approx 50µl) so that each section is covered. Incubate for 10 minutes.
5) Wash well in PBS.
6) Apply streptavidin alkaline phosphatase (red solution) to both sections. This solution does not need to be diluted. Apply (approx 50µl) so that each section is covered. Incubate for 10 minutes.
7) Wash well in PBS.
8) Apply chromogen-substrate solution (BCIP/NBT) to both sections. This solution does not need to be diluted. Apply (approx 50µl) so that each section is covered. Incubate for 15 minutes. During this time you should wash your sections very carefully for signs of the colour reaction. If your sections begin to turn dark there may be background colour developing and the solution should be removed.
9) Wash well in PBS.
10) Counterstain nuclei with Haematoxylin or Nuclear Fast Red. Stain by placing the sections in each of the coplin jars provided. (Nuclear stain – H20wash – dehydration series: 70%, 96%, 96%, 100%, 100% EtOH- histoclear). BEWARE: when clearing slides for permanent mounting do not use zylene as this will cause staining to be lost.
11) Mount and coverslip sections and view under light microscope. BEWARE: The mounting media will still be wet, so be careful not to touch the coverslips with the objective lens.
Questions:
1) Name the 4 components of the LSAB method used in this practical:
[pic]
2) Outline at least 3 advantages of this staining method:
3) Comment on the appearance and any differences between the test and control (no primary antibody) sections. Sketch the appearance of the test section:
4) Explain why the control section (no primary antibody) is useful:
5) Briefly describe two other controls (positive or negative) that could have been carried out to further validate this method:
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