270 ORIGIN OF AMPHIBIAN PIGMENT 



SELECTIVE STAINING OF IN VIVO NEURAL CREST DERIVATIVES 



In transplantation and isolation experiments there is always the question as to 

 whether the derivatives of the excised anlage are the sajne as they would have been in the 

 original site of the donor. Stone (1952) refined a method of preserving the vital dye 

 Nile Blue Sulphate in the fixed emhryo so that it could be located in sectioned material. 

 It is therefore in the nature of a confirmatory experiment that the following procedure 

 1b given. 



1. Select Urodele embryos of stage #25, and remove all of their membranes in Growing 

 Medium. Place them in an operating dish with a Permoplast or soft paraffin base, 

 and mould depressions to hold them with the neural folds uppermost. 



2. Cut a piece of Nile Blue dyed agar (0.1 gr. Nile Blue Sulphate in 2^ agar in 100 

 cc. of distilled water, dissolved by heating-, and poured, while hot, onto glass 

 plates covered with a very thin layer of glycerine. When dried, the agar can be 

 pealed off of the plate In thin sheets of any size or shape (e.g., the shape and 

 size of the neural fold). Place this minute piece of agar flat on a piece of 

 coverslip and pass the coverslip through a flame. This will cause the agar to 

 melt slightly, and become adherent to the coverslip chip. With practice one can 

 provide a marker of the exact shape and size of the neural fold. 



5. After the neurula stage (#25) is firmly placed within the depression. In Standard 

 Solution, bring the (Inverted) coverslip chip into position so that the Nile Blue 

 Agar will make precise contact with one of the neural folds. Press it against the 

 embryo, and anchor the edges of the glass chip in the surrounding Permoplast. 

 Hold the neurula in this position for 20-50 minutes, while the dye is being trans- 

 ferred to the neural fold, and then gently remove it without tearing away any of 

 the cells of the embryo. 



k. Transfer the embryo to Urodele Growing Medium for development until stage #28 or 

 later. The dye will remain for a long time and spread with the cells It has In- 

 vaded, so that It may be found even after the pigment has begun to appear. 



5. (a) Fix the embryo in Zenker-acetic for two hours; wash in running tap water 

 1 hour. 



(b) Place in 1^ aqueous solution of phosphomolybdlc acid for 2 hours (Lehmann, 

 1929). 



(c) Transfer for half hour periods through the ascending alcohols to each of which 

 has been added 0.1^ phosphomolybdlc acid. This acid preserves the dye. 



(d) Transfer for one-half hour to a mixture of equal parts of 100^ alcohol, (con- 

 taining 0.1^ phosphomolybdlc acid) and cedar oil. Then place in pure cedar 

 oil until clear (overnight). 



(e) Embed in three changes of paraffin-Bayberry-beeswax mixture (90 - 5 - 5) and, 

 when hardened, section at 10 microns. The sections may be treated in the usual 

 way (with xylol) and mounted under clarite. If the tissues are not passed 

 through any water they will retain the Nile Blue Sulphate dye in the cells 

 which were originally stained, and with appropriate lighting the demarcation 

 between stained eind unstained areas can be made out easily. 



Becord on following page by drawings or photographs the results of in vivo staining 

 of the neural crest anlage. 



OBSERVATIONS AND TABULATION OF DATA: 



The data for these experiments are qualitative rather than quantitative, and space is 

 provided with each section for appropriate records. Histological sections, when possible, 

 will confirm the macroscopic analyses. (See DuShane, 1955= Jour. Exp. Zool. 72:1 for 

 cytologlcal procedures relative to the various types of chromatophores. ) 



