Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
This unit describes selected methods for fixing and sectioning various forms of biological materials ranging from tissues to single cells. Sections prepared according to these protocols can then be used to examine cell and tissue morphology and in studies involving in situ hybridization, immunohistochemistry, and enzyme histochemistry. The basic protocol describes how tissues and embryos can be prepared for sectioning by fixing in paraformaldehyde followed by embedding in wax, while the alternate protocol describes fixation of suspended or cultured cells. Two support protocols cover preserving and fixing organs by perfusion of whole animals with paraformaldehyde and a procedure for sectioning wax blocks of fixed tissue, plus the subsequent mounting of sections onto prepared or “subbed” glass slides.
Table of Contents
- Basic Protocol 1: Paraformaldehyde Fixation and Paraffin Wax Embedding of Tissues and Embryos
- Alternate Protocol 1: Fixation of Suspended and Cultured Cells
- Support Protocol 1: Perfusion of Adult Mice
- Support Protocol 2: Sectioning Samples in Wax Blocks
- Reagents and Solutions
- Literature Cited
Basic Protocol 1: Paraformaldehyde Fixation and Paraffin Wax Embedding of Tissues and Embryos
Alternate Protocol 1: Fixation of Suspended and Cultured Cells
Support Protocol 1: Perfusion of Adult Mice
Support Protocol 2: Sectioning Samples in Wax Blocks
Figure 14.1.1 Perfusion of a mouse. This diagram illustrates the chambers of the heart and the correct positioning of the syringe in the left ventricle for perfusion.
Figure 14.1.2 Placement of paraffin sections on gelatin‐subbed glass slides. Steps A to E depicted here correspond to steps to in the support protocol.
|Berger, C.N. 1986. In situ hybridization of immunoglobulin‐specific RNA in single cells of the β‐lymphocyte lineage with radiolabeled DNA probes. EMBO J. 5:85‐93.|
|Luna, L.G. 1968. Manual of Histologic Staining: Methods of the Armed Forces Institute of Pathology (3rd ed.). McGraw‐Hill, New York, N.Y.|
|Muller, W.J., Sinn, E., Pattengale, P.K., Wallace, R., and Leder, P. 1988. Single‐step induction of mammary adenocarcinoma in transgenic mice bearing the activated c‐neu oncogene. Cell 54:105‐115.|
|Zeller, R., Bloch, K.D., Williams, B.S., Arceci, R.J., and Seidman, C.E. 1987. Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev. 1:693‐698.|