IntroductionAntibodies have highly specific binding sights which bind to complex molecules such as proteins and glycoproteins. In this study, antibodies that recognize actin were used to study the localization of actin in mouse lung tissues. Beta actin was the primary isoform of interest, although alpha and gamma isoforms were also detected using an anti-pan actin antibody.

Beta actin is a cytoskeletal protein involved in cell motility. Cell motility is normal in white blood cells and during embryonic development; however, aberrant tumor cells are capable of movement in the process of metastasis (Pollard and Cooper, 2009, 1210). Metastasis is the movement of cancer cells from the primary tumor to a distant location in another tissue type (Weiss and Ward, 1983, 112; Horak and Steeg, 2005, 93; Lambrechts et al., 2004, 1900). The most common form of lung cancer originates in the bronchiole epithelium and is highly metastatic (Anonymous, 2010). The study of beta-actin in lung cells may help in the development of new cancer treatments.

In this investigation it was hypothesized that anti-pan-actin antibodies would stain in all cell types, particularly muscle tissue due to the large amount of alpha-actin. Anti-beta-actin antibodies were hypothesized to stain in all cell types although much more weakly than the anti-pan-actin antibodies. Healthy lung tissues were used in this study, although cancerous lung tissues would be of interest in futur investigations.

Materials and MethodsMouse lungs were resected and fixed in Histochoice fixative. The lungs were then serially dehydrated and embedded in paraffin. Tissues were sectioned to widths of 4-10 μm. Sections were incubated with mouse anti-beta actin primary antibodies and goat anti-mouse IgG actin secondary antibodies for the localization of beta-actin isoform. Alternatively, sections were stained with rabbit anti-pan-actin primary antibodies and goat anti-rabbit IgG secondary antibodies for staining of all actin isoforms. Hematoxylin and eosin stains were used for anatomical imaging.

ResultsAnti-pan-actin antibodies stained all tissues, particularly muscle tissues. However, the overall antibody reaction was weak. Anti-beta-actin

antibodies appeared to specifically stain all cell types with the heaviest staining being in bronchiole epithelial cells. However,

the control lacking primary antibodies revealed that the secondary antibodies were staining the tissues non-

specifically.

Distribution of Actin Isoforms by Immunohistochemistry in Mouse Lung TissueJenny Bruning, Marietta College Advisor: Dr. Steve Spilatro

Figure 1

Figure 1. H and E staining of lung tissues. A. Lung section. 1. Bronchiole leading into alveolar ducts. 2. Blood vessel. 3. Bronchus. B. Bronchiole epithelium cross section. Scale bar represents 100 μm.

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Figure 2

Figure 2. A and B stained with rabbit anti-beta-actin primary antibody and goat anti-rabbit IgG secondary antibody. Specific staining in all tissues, strong staining in muscle cells. C. Control tissue incubated with no primary antibodies and goat anti-rabbit IgG, no specific staining present. D. Control tissue incubated with rabbit anti-beta-actin primary antibody and no secondary antibody. No specific staining present.

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ConclusionsThe hypothesis stated that both anti-pan and anti-beta-actin antibodies would stain all cell types with anti-pan-actin staining muscle cells more intensely and anti-beta actin antibodies staining all cells strongly. Staining of all actin isoforms using anti-pan-actin antibodies revealed specific staining of all cell types with the most intense staining being in muscle tissues, supporting the hypothesis.

Goat anti-mouse IgG secondary antibodies used in detection of mouse-anti-beta-actin primary antibodies bound non-specifically in the mouse lung tissues. It was concluded that the goat anti-mouse IgG antibodies were binding to mouse IgGs initially present in the mouse lung tissues. During preparation of the methods the type of anti-IgG secondary antibodies were not considered. For successful beta-actin probing, primary antibodies raised in mammals other than mice must be used. Therefore, all beta-actin results must be discarded.

Literature CitedAnonymous. What are the key statistics about lung cancer? [Internet]. American Cancer Society. [revised 2010 December 16; cited 2011 January 12]. http://www.cancer.org/Cancer/LungCancer-Non-SmallCell/DetailedGuide/non-small-cell-lung-cancer-key-statistics.Horak CE, Steeg PS. 2005. Metastasis Gets Site Specific. Cancer Cell 8(2): 93-95.Kumar V, Cotran RS, Robbins SL. 2003. Robbins Basic Pathology 7 th ed. Philadelphia (PA): Saunders; 873p.Lambrechts A, Van Troys M, Ampe C. 2004. The actin cytoskeleton in normal and pathological cell motility. The International Journal of Biochemistry and Cell Biology 36: 1890-1909.Nowak D, Skarek-Maruszewska A, Zemanek-Zboch M, Malicka-Blaszkiewicz M. 2005. Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential. Acta Biochimica Polonica 52(2):461-468.Pollard TD, Cooper JA. 2009. Actin, a Central Player in Cell Shape and Movement. Science 326: 1208-1212.Weiss L, Ward PM. 1983. Cell detachment and metastasis. Cancer and Metastasis Review 2(2): 111-127.

AcknowledgementsSpecial thanks to my advisor, Dr. Steve Spilatro, for helpng me through every part of this process. Also thanks to Dr. David Brown for instructing the 2011 Bio 490 capstone class.

Figure 3

Figure 3. A. Tissue section incubated with mouse anti-beta actin antibodies and goat anti-mouse-IgG secondary antibodies. Staining in all cell types. B. Control section incubated with goat anti-mouse IgG secondary antibodies with no primary antibodies. Nonspecific staining occurred in all cell types. C. Control section incubated with mouse anti-beta-actin primary antibody and no secondary antibodies. No staining visible. Scale bars represent 100 μm.

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