Double staining method

Certain guidelines (5-7) require or give as an option a double staining method for the fetal skeleton with Alcian Blue added to stain cartilage together with Alizarin for the ossified material. 

Kazama, J. J., Aikata, T., Arakawa, M., and Ozawa, H. (1994) A new histochemical double stain method using three-dimensional analysis with confocal laser scanning microscopy. Biotech. Histochem. 69, 324-328. 

Figure 1. Sequential double staining method performed with the EnVision G 2 Doublestain Kit using polyclonal anti-kappa light chains (red) and polyclonal anti-lambda light chains (brown) as primary antibodies. Formalin-fixed, paraffin-embedded tissue sections from tonsils.

A satisfactory "sequential" double-staining method using mouse monoclonal antibodies has been in routine use in our laboratory for some time. The method is applicable both to frozen sections and to cytocentrifuge preparations. 

Van der Loos CM, Gobel H (2000) The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens. J Histochem Cytochem 48 1431-1437... 

Saito, N. Double staining method and enzyme reaction chamber for protoplasts preparation. Jpn. Kokai Tokkyo Koho JP 06102158,1994. 

A collection of useful protocols for the simultaneous immunoenzyme staining of two or more tissue antigens is available on the websites http //www.protocol-online.org/prot/Immunology/ http //www.vectorlabs.com/Protocols/MLB.pdf and http //www.ihcworld.com/. The protocols given in this chapter are practiced in the authors laboratory. These methods fall into two main categories (a) simultaneous immunoenzymatic double staining, and (b) sequential immunoenzymatic double/ multiple staining. 

Zebhe, L, Sallstrom, J. F., Hacker, G. W., Hauser-Kronberger, C., Rylander, E., and Wilander, E. (1994) Indirect and direct on situ PCR for the detection of human papillomavirus. An evaluation of two methods and a double staining technique. Cell Vision 1, 163-167. 

Prenatal developmental toxicity testing of chemicals and pesticides requires evaluation of both cartilaginous and ossified skeletal components, but the corresponding testing guidelines do not specify how. Double staining is the preferred method and is strongly recommended. 

Peters PWJ (1977) Double staining of fetal skeletons for cartilage and bone. In Neubert D, Merker HJ, Kwasigroch TE (eds) Methods in prenatal toxicology. George Thieme, Smttgart, pp 153-154... 

Simultaneous staining In a simultaneous double stain, the primary antibodies can be applied simultaneously. The advantage of this method is that it is less time-consuming because the reagents can be mixed together. However, the technique can only be used, if suitable primary antibodies are available. Two methods can be adopted A direct method with directly-labeled primary antibodies, or an indirect method based on unlabeled primary antibodies raised in different host species, or of different Ig isotype or IgG subclass (4). 

One good method of detection on TLC plates is the use of iodine. After the plate has been run and dried, it is placed in a closed container containing iodine. The iodine vapor reacts with double bonds in the compounds to form brown complexes. If iodine doesn t work, then other staining methods will have to be examined6 (ideally the staining method should be reversible so that the compound identity can be confirmed by HPLC after the plate has been run and stained). 

Fig. 6. Cellular colocalaation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the neostriatum. Each number indicates the same glial cell showing both GDH and GS immunoreactivities. The figure is modified from Kaneko et al. (1988a), where the method for double staining is described. Briefly, GS was immunolabeled with anti-GS rabbit serum and fluorescein-labeled anti-rabbit IgG antibody, and after blocking the sections with normal rabbit serum GDH was visualized by the immunoperoxidase method with biotinylated anti-GDH rabbit IgG and ABC.

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