Selective impregnation of developing neurons with the Loyez method and with two kinds of Luxol Fast Blue

In chick embryos, unlike in adults, many neurons can be impregnated selectively with the LOYEZ iron-haematoxylin method, and with the MBSN and ARN types of Luxol Fast Blue. The same neurons are selected with each of the 3 methods. Their distribution at a given age is roughly predictable, but it changes with age, as is described in some detail. Large neurons are impregnated more readily than small neurons; glial cells are not impregnated. With each of the 3 methods, the same conditions of fixation and embedding are needed for the selective impregnation to occur: fixation with a dilute, alkaline solution of formaldehyde (or, non-optimally, a closely related aldehyde), and nitrocellulose embedding. Numerous other stains fail to produce the selective impregnation. The parameters of the LOYEZ method have been varied systematically in order to optimize the neuronal impregnation, and preliminary efforts have been made to deduce the substrate of the staining, which probably involves proteins or peptides.