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A Simple Silver-Gold Intensification Procedure for Double DAB Labeling Studies in Electron Microscopy

1997; SAGE Publishing; Volume: 45; Issue: 4 Linguagem: Inglês

10.1177/002215549704500414

1551-5044

Rebecca Teclemariam‐Mesbah, Joke Wortel, Herms J. Romijn, Ruud M. Buijs,

Health, Environment, Cognitive Aging

Pre-embedding intensification procedures for double labeling in electron microscopy available to us (Gorcs et al. 1986; Van den Pol and Gorcs 1986; Liposits et al. 1984) are laborious and have many drawbacks, such as a few hours preincubation with thioglycolic acid (Gallyas 1982), which not only lengthens the intensification procedure but also in some cases makes it impossible to perform a second immunostaining (Buijs et al. 1990). Second, any chemical residues on the glassware or pipet influence the final outcome of the silver reaction, which is therefore difficult to control accurately. To overcome these drawbacks, we developed a methenamine silver–gold method for DAB labeling studies that allows reproducible intensification for light and electron microscopy, modified from the technique described by Rodriguez et al. (1984). Fiftym m-thick sections of brains fixed with 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) were treated with freshly made 0.5% sodium borohydride (NaBH 4 ) in Tris-buffered saline (TBS) for 10 min. This pretreatment decreased the background staining tremendously but without affecting the tissue structure. The sections were then rinsed several times in TBS until they sank. Then the sections were incubated in 50% ethanol for 30 min to increase antibody penetration, after which they were processed for the first immunostaining until they were allowed to react with 0.025% 3,3 9 -diaminobenzidine tetrahydrochloride (DAB) in the presence of 0.006% H 2 O 2 for 15 min. DAB staining was then intensified by methenamine silver-gold reaction as follows.