An immunohistochemical method for the detection of proteins in the vertebrate lens

doi:10.1016/S0022-1759(01)00374-X

Journal of Immunological Methods

Volume 253, Issues 1–2, 1 July 2001, Pages 243-252

https://doi.org/10.1016/S0022-1759(01)00374-X Get rights and content

Abstract

Fluorescence immunohistochemistry has traditionally been difficult or impossible to perform on the vertebrate lens because of its extremely high protein content. Described here is a robust and rapid method for preparing and labeling vertebrate eyes for confocal microscopy. This technique has successfully been applied to localize proteins in the lens epithelium and capsule, as well as the primary and secondary fibers. This technique preserves tissue morphology and coupled with double and triple labeling, has allowed localization of proteins bound to plasma membrane, basement membrane, nucleus, endoplasmic reticulum as well as sub-nuclear compartments. In addition, the present technique has proven useful for fluorescent immunohistochemical analysis of diverse tissues including whole embryos, adult muscle, pancreas, and liver. This procedure allowed us to successfully localize a wide variety of antigens on diverse vertebrate tissues including the more challenging vertebrate lens.

Section snippets

Type of research

1.1. Duncan, M.K., Cvekl, A., Li, X., Piatigorsky, J., 2000. Truncated forms of Pax-6 disrupt lens morphology in transgenic mice. Invest. Ophthalmol. Vis. Sci., 41, 464.

1.2. Ma, H., Shih, M., Fukiage, C., Azuma, M., Duncan, M.K., Reed, N.A., Richard, I., Beckmann, J.S., Shearer, T.R., 2000. Influence of specific regions in Lp82 calpain on protein stability, activity, and localization within lens. Invest. Ophthalmol. Vis. Sci., 41, 4232.

1.3. Duncan, M.K., Kozmik, Z., Cveklova, K., Piatigorsky,

Time required

2.1. Tissue dissection, embedment and sectioning: time variable.

2.2. Fix tissue in acetone:methanol: 10 min.

2.3. Allow samples to air dry: 10 min.

2.4. Block non-specific protein binding sites on slides and sections with 1% BSA: 60 min.

2.5. Incubate with primary antibody: 60 min.

2.6. Wash slides with PBS: 30 min.

2.7. Incubate with secondary antibody and counterstain: 60 min.

2.8. Wash slides with PBS: 30 min.

2.9. Add mounting media and coverslip: 90 s per slide.

Total estimated time for labeling

Special equipment

3.1.1. Tissue sections were obtained with a Leica CM 3050 S Cryostat with CE knife holder, specimen temperature control, and motorized sectioning. Leica Microsystems, 111 Deer Lake Road, Deer Field, IL 60015.

3.1.2. Images were obtained using a Zeiss LSM 510 Confocal Microscope configured with an Argon/Krypton laser (488 and 568 nm excitation lines) and Helium Neon laser (633 nm excitation line). Carl Zeiss, Königsallee, 9-21 37081, Göttingen, Germany.

Tissue sectioning materials

3.2.1. Twenty Slide Tray Holder. Fisher

Embedding and sectioning

4.1.1. Fill specimen mold with Tissue Freezing Media (TFM), avoiding introduction of air bubbles.

4.1.2. Place freshly excised tissue into mold oriented as shown in Fig. 1a. It is important to place tissue as close as possible to side 1 to minimize sectioning and as far as possible from side 2 and 4 to avoid tearing of the tissue by the knife. It is helpful to perform this step under a dissecting microscope.

4.1.3. Once tissue is properly oriented, place mold on block of dry ice to freeze

Results

Fluorescent immunohistochemical labeling of the adult vertebrate lens has been problematic due to difficulties in preparing high quality 6–8 μm sections because of the physical properties of the lens. These problems include poor fixative penetration in unsectioned tissue, preparation of sections with acceptable morphology, non-specific binding of antibodies due to high protein concentration within the lens, and light scatter/autofluorescence induced by lens proteins. Thus, most reports of

Discussion

The following is a list of the steps/precautions that are necessary to optimize the best immunohistochemical labeling results.

Essential literature references

Francis, P.J., Berry, V., Moore, A.T., Bhattacharya, S., 1999. Lens biology: development and human cataractogenesis. Trends Genet. 15, 191 (Review).

Harlow, E., Lane, D., 1998. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York, NY.

Jean, D., Ewan, K., Gruss, P., 1998. Molecular regulators involved in vertebrate eye development. Mech. Dev. 76 (1–2) 3 (Review).

Oliver, G., Gruss, P., 1997. Current views on eye development. Trends Neurosci. 20, 415 (Review).

Oyster, C.W., 1999.

Detailed procedure of immunohistochemical staining

8.1.1. Select slides from the −80°C.

8.1.2. Immediately place frozen slides into a coplin jar or a slide dish filled with 1:1 acetone:methanol pre-chilled to −20°C and incubate for 10 min or longer at −20°C.

8.1.4. Remove sections from −20°C and allow to air dry in the slide rack.

8.1.7. Overlay tissue with 1% BSA in 1×PBS and block for 60 min at RT.

8.1.9. Gently shake off blocking solution and overlay tissue with 75 μl of diluted primary antibody in blocking buffer. Incubate at RT for 60 min.

Acknowledgements

The authors would like to thank Paula Ousley of the Oregon Lions Eye Bank, and Patrick Kelley, of the University of Delaware, for technical assistance, Johnell Cheong for scientific artwork, as well as, Joanne Julian, University of Delaware, and Steve Bassnett, Washington University, for invaluable advice early in the development of this protocol.

References (8)

M.K. Duncan et al.
Developmental regulation of the chicken beta B1-crystallin promoter in transgenic mice
Mech. Dev.
(1996)
S. Bassnett et al.
Chromatin degradation in differentiating fiber cells of the eye lens
J. Cell. Biol.
(1997)
J.P. Brady et al.
Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin
Proc. Natl. Acad. Sci.
(1997)
M.K. Duncan et al.
Truncated forms of Pax-6 disrupt lens morphology in transgenic mice
Invest. Ophthalmol. Vis. Sci.
(2000)

There are more references available in the full text version of this article.

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^☆: Supported in part by National Institutes of Health Grants EY12221 (MKD) and IDEA RR11820 (Department of Biological Sciences, University of Delaware)

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ProtocolAn immunohistochemical method for the detection of proteins in the vertebrate lens☆

Abstract

Section snippets

Type of research

Time required

Special equipment

Tissue sectioning materials

Embedding and sectioning

Results

Discussion

Essential literature references

Detailed procedure of immunohistochemical staining

Acknowledgements

Mech. Dev.

Chromatin degradation in differentiating fiber cells of the eye lens

J. Cell. Biol.

Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin

Proc. Natl. Acad. Sci.

Truncated forms of Pax-6 disrupt lens morphology in transgenic mice

Invest. Ophthalmol. Vis. Sci.

Protocol
An immunohistochemical method for the detection of proteins in the vertebrate lens☆