Staining methods immunocytochemistry

Plant Cells and Tissues Structure-Function Relationships. Methods for the Cytochemical/Histochemical Localization of Plant Cell/Tissue Chemicals. Methods in Light Microscope Radioautography. Some Fluorescence Microscopical Methods for Use with Algal, Fungal, and Plant Cells. Fluorescence Microscopy of Aniline Blue Stained Pistils. A Short Introduction to Immunocytochemistry and a Protocol for Immunovi-sualization of Proteins with Alkaline Phosphatase. The Fixation of Chemical Forms on Nitrocellulose Membranes. Dark-Field Microscopy and Its Application to Pollen Tube Culture. Computer-Assisted Microphotometry. Isolation and Characterization of... 

Gnstafsson, M. K. (1991) Skin the tapeworms before you stain their nervous system A new method for whole-mount immunocytochemistry. Parasitology Res. 77, 509-516. 

ELISA), radioimmunoadsorbent assays (RIA), Western blotting, or other techniques (17,22-24). These methods will detect the presence and also to some extent the specificity of a particular antibody, but will not ensure that the antibody is also suitable for immunocytochemistry (25). For this reason, the antibody should be tested under the experimental conditions of fixing, embedding, and staining, and on the desired tissue to be used subsequently. 

Nucleic acid immunocytochemistry also differs from nucleic acid cytochemical approaches in that, for the latter, a reagent, such as a stain, reacts directly with the cellular nucleic acids. Such methods lack the exquisite specificity and sensitivity of nucleic acid immunocytochemistry. Table 1 compares these methods. 

Fig. 9. Calcium binding proteins in the MOB. A. Distribution of cells labeled with an antibody to Calbindin (CALB) using immunocytochemistry. B. Parvalbumin (PARV) immunocytochemically labeled cells are found mainly in the EPL in the bulb. C. Golgi stained section showing impregnated external tufted cells. D. An example of how cells may be identified by other methods. In this case an external tufted cell is shown after intracellular injection of the marker biocytin. Bar in D, /tm.

It is possible to use HRP as a label for electron microscopic immunocytochemistry because the DAB-developed chromogen generates a reaction product that is easily stained with heavy metals. While HRP can be used for electron microscopic immunocytochemistry, the DAB reaction product does not show high-resolution distribution of the antigen labeled by the 1° antibody. The HRP method has been used to fill specific neurons and trace their dendrites to determine types of synaptic contacts. However, the diffused nature of the reaction product does not allow it to be associated with specific cellular organelles. Therefore, using HRP for electron microscopic immunocytochemistry will not be discussed here. 

Burry RW (1995) Pre-embedding immunocytochemistry with silver-enhanced small gold particles. In Hayat MA (ed.) Immunogold-sUver staining principles, methods and applications. Boca Raton, FL CRC Press, pp 217-230. 

More recently we have investigated whether the ubiquitin-proteosome system is perturbed in the heart of human DCM patients (Weekes et al., 2003). As in bovine DCM, expression of the enzyme UCH was more than 8-fold elevated at the protein level and more than 5-fold elevated at the mRNA level in human DCM. Moreover, this increased expression of UCH was shown by immunocytochemistry to be associated with the myocytes which do not exhibit detectable staining in control hearts. Overall protein ubiquitination was increased 5-fold in DCM relative to control hearts and using a selective affinity purification method we were able to demonstrate enhanced ubiquitination of a number of distinct proteins in DCM hearts. We have identified a number of these proteins by mass spectrometry. Interestingly many of these proteins were the same proteins that we have previously found to be present at reduced abundance in DCM hearts (Corbett et al., 1998). This new evidence strengthens our hypothesis that inappropriate ubiquitin conjugation leads to proteolysis and depletion of certain proteins in the DCM heart and may contribute to loss of normal cellular function in the diseased heart. 

---