Pre-embedding staining

Almost all pre-embedding staining procedures consist in the following steps (i) prefixation to obtain maximum permeabilization of the tissue with the best acceptable structural preservation and minimal loss of antigens (ii) immunohistochemical incubations (iii) further fixation (iv) revelation of the enzyme (v) postfixation (e.g., with osmic acid to render the DAB product electron dense) and, (vi) dehydration and embedding. Extensive washings are included between the various steps. The enzyme-generated product should be insoluble, both in water and alcohol. 

Fig. 4. Cervical dorsal root ganglia immunolabeled (pre-embedding immunoperoxidase) for GluR2/3 (a) and GluR4 (b). Arrows indicate satellite cells showing little or no immunolabeling with antibody to GluR2/3 and stained densely with antibody to GluR4. From Tachibana et al. (1994), reproduced with permission from Wiley-Liss, Inc.

The dehydration, embedding, and sectioning of pre-embedding electron microscopic immunocytochemistry blocks follow the standard electron microscopic procedures. However, for section staining, the time is reduced to 2-3 min to preserve the size of the silver particles. 

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. 

Burry. R. W. (1995). Pre-embedding immunsilver-enhanced small gold particles. In "Immunogold-Silver Staining Principles. Methods, and Applications" (M. A. Hayat. cd.), pp. 217-230. CRC Press, Boca Raton. FL. 

Figure 21.5. Human pre-adipocyte (derived fromfresh human bone marrow) culture inside mineralized chitosan-coated alginate cap>sules (n=12). A) A single whole capsule containing 450,000 pre-adipocytes at 24 hours. Pre-adipocyte cells are metabolically very active and exapnd in numbers rapidly after encapsulation. B) Embedded pre-adipocytes produce rounded colonies containing dense oil droplet formations in complete (+single insulin dose) media at day 21. C) Oil droplets stained with oil red-O to indicate presence of fat inside observed droplets at day 12. D) Oil-droplet formation from same population of cells grown in 2D mono-layer culture at day 21.

---