Hostname: page-component-76fb5796d-vvkck Total loading time: 0 Render date: 2024-04-26T07:43:07.749Z Has data issue: false hasContentIssue false
  • English
  • Français

Immunolocalization of MAP-2 in Routinely Formalin-Fixed, Paraffin-Embedded Guinea Pig Brain Sections Using Microwave Irradiation: A Comparison of Different Combinations of Antibody Clones and Antigen Retrieval Buffer Solutions

Published online by Cambridge University Press:  08 March 2005

Robert K. Kan ,
Christina M. Pleva ,
Tracey A. Hamilton  and
John P. Petrali
Robert K. Kan
Affiliation:
Comparative Pathology Branch, Comparative Medicine Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA
Christina M. Pleva
Affiliation:
Comparative Pathology Branch, Comparative Medicine Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA
Tracey A. Hamilton
Affiliation:
Comparative Pathology Branch, Comparative Medicine Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA
John P. Petrali
Affiliation:
Comparative Pathology Branch, Comparative Medicine Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA
Get access

Abstract

The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.

Keywords

antigen retrievalimmunohistochemistrycytoskeletal proteinformaldehydefixationpretreatmentnerve agentsoman
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 11 , Issue 2 , April 2005 , pp. 175 - 180
Copyright
© 2005 Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Ballough, G.P., Martin, L.J., Cann, F.J., Graham, J.S., Smith, C.D., Kling, C.E., Forster, J.S., Phann, S., & Filbert, M.G. (1995). Microtubule-associated protein 2 (MAP-2): A sensitive marker of seizure-related brain damage. J Neurosci Methods 61, 23–32.Google Scholar
Caceres, A., Banker, G., Steward, O., Binder, L., & Payne, M. (1984). MAP2 is localized to the dendrites of hippocampal neurons which develop in culture. Brain Res 315, 314–318.Google Scholar
De Camilli, P., Miller, P.E., Navone, F., Theurkauf, W.E., & Vallee, R.B. (1984). Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience 11, 817–846.Google Scholar
Evers, P. & Uylings, H.B. (1994). Microwave-stimulated antigen retrieval is pH and temperature dependent. J Histochem Cytochem 42, 1555–1563.Google Scholar
Evers, P. & Uylings, H.B. (1997). An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. J Neurosci Methods 72, 197–207.Google Scholar
Folkerts, M.M., Berman, R.F., Muizelaar, J.P., & Rafols, J.A. (1998). Disruption of MAP-2 immunostaining in rat hippocampus after traumatic brain injury. J Neurotrauma 15, 349–363.Google Scholar
Fox, C.H., Johnson, F.B., Whiting, J., & Roller, P.P. (1985). Formaldehyde fixation. J Histochem Cytochem 33, 845–853.Google Scholar
Gown, A.M., Wever, N., & Battifora, H. (1993). Microwave-based antigenic unmasking: A revolutionary new technique for routine immunohistochemistry. Appl Immunhistochem 1, 256–266.Google Scholar
Hazelbag, H.M., van den Broek, L.J., van Dorst, E.B., Offerhaus, G.J., Fleuren, G.J., & Hogendoorn, P.C. (1995). Immunostaining of chain-specific keratins on formalin-fixed, paraffin-embedded tissues: A comparison of various antigen retrieval systems using microwave heating and proteolytic pre-treatments. J Histochem Cytochem 43, 429–437.Google Scholar
Hsu, S.M., Raine, L., & Fanger, H. (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29, 577–580.Google Scholar
Kitagawa, K., Matsumoto, M., Niinobe, M., Mikoshiba, K., Hata, R., Ueda, H., Handa, N., Fukunaga, R., Isaka, Y., & Kimura, K. (1989). Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage—Immunohistochemical investigation of dendritic damage. Neuroscience 31, 401–411.Google Scholar
Matesic, D.F. & Lin, R.C. (1994). Microtubule-associated protein 2 as an early indicator of ischemia-induced neurodegeneration in the gerbil forebrain. J Neurochem 63, 1012–1020.Google Scholar
Pileri, S.A., Roncador, G., Ceccarelli, C., Piccioli, M., Briskomatis, A., Sabattini, E., Ascani, S., Santini, D., Piccaluga, P.P., Leone, O., Damiani, S., Ercolessi, C., Sandri, F., Pieri, F., Leoncini, L., & Falini, B. (1997). Antigen retrieval techniques in immunohistochemistry: Comparison of different methods. J Pathol 183, 116–123.Google Scholar
Posmantur, R.M., Kampfl, A., Taft, W.C., Bhattacharjee, M., Dixon, C.E., Bao, J., & Hayes, R.L. (1996). Diminished microtubule-associated protein 2 (MAP2) immunoreactivity following cortical impact brain injury. J Neurotrauma 13, 125–137.Google Scholar
Shi, S.R., Imam, S.A., Young, L., Cote, R.J., & Taylor, C.R. (1995). Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43, 193–201.Google Scholar
Shi, S.R., Key, M.E., & Kalra, K.L. (1991). Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39, 741–748.Google Scholar
Shiurba, R.A., Spooner, E.T., Ishiguro, K., Takahashi, M., Yoshida, R., Wheelock, T.R., Imahori, K., Cataldo, A.M., & Nixon, R.A. (1998). Immunocytochemistry of formalin-fixed human brain tissues: Microwave irradiation of free-floating sections. Brain Res Brain Res Protoc 2, 109–119.Google Scholar