XX CHOICE OF METHODS 



After fixation some Washing of the tissue in water is necessary unless it has 

 been fixed in alcohol, Carnoy or similar mixtures. The next step is Dehydration 

 and a choice must be made between slow and rapid methods. Sometimes a 

 substitute for alcohol is indicated. If Imbedding is to be in celloidin Clearing 

 in a xylol-like fluid is omitted and heating is unnecessary. There are many ways 

 of clearing preliminary to paraffin imbedding. In Sectioning the thickness 

 depends upon the purpose in view. Thick sections may be as necessary as thin 

 ones and serial sections are often required. In the Mounting of sections on 

 slides the use of water must occasionally be avoided. Numerous techniques are 

 applicable to the sections and are given individually later either under the head- 

 ing of the substance or structure to be demonstrated or under the name of the 

 technique or its introducer. For choice see Staining. 



Many beautifully stained sections of well fixed tissue are of but little value, 

 because the investigator failed to note the exact location in the organ or tissue 

 whence they were excised and omitted to have the sections cut in the most 

 favorable plane. 



5. To Mark Selected Individual Cells or Tissues in Vivo 



FOR Later Examination 



In this connection we at once think of the vital stains, trypan blue, carmine, 

 India ink (carbon) and hundreds of others, which, when injected into the body, 

 are phagocytosed by the reticulo-endothelial cells (or macrophages). Pieces of 

 tissue can then be excised and the accumulations of stains can be studied within 

 the still living cells, that is supravitally, for unless cultured the cells are slowly 

 dying. But, if desired, the tissues can be fixed and the results observed at 

 leisure in sections. 



It has long been known that bone laid down in the presence of Madder fed to 

 the animals is marked by the madder and can thus be distinguished from bone 

 deposited beforehand and afterwards. In the same way dentine can be marked 

 in vivo with Alizarin Red S. 



Another example of in vivo marking is the deposition of Prussian Blue. Thus 

 a slightly hypertonic solution (potassium ferrocyanide 0.5 gm., iron ammonium 

 citrate, 0.5 gm. and aq. dest. 50 cc.) injected into the subarachnoid space of the 

 spinal cord is useful in the localization of the pathways of drainage of cerebro- 

 spinal fluid, because of the marking secured when the tissues are fixed in 40% 

 formalin plus 1% concentrated hydrochloric acid by the deposition of Prussian 

 blue (Weed, L. H., J. Med. Res., 1914, 26, 21-117). 



The tissues of animals recently killed or under anesthesia can be selectively 

 marked with various dyes by Perfusion of the blood vessels with dilute solutions 

 of dyes. The outstanding methods in this group have been devised by Bensley 

 (R. R., Am. J. Anat., 1911, 12, 297-388) for histological analysis of the epithelial 

 components of the pancreas and stomach. Dilute solutions of the dyes in physio- 

 logical saline are injected into the thoracic aorta of an animal killed by bleeding. 

 Pieces of pancreas and gastric mucous membrane are then removed and examined 



