There are very few, practically no, detailed histochemical studies on the distribution of phosphatases in the rat skin through its organogenesis from embryonic stage, although there have been several fairly extensive studies on the distribution of the phosphatases in the adult human and animal skins (Montagna, '47; Montagna and Hamilton, '47; Fisher and Glick, '47; Spier and Martin, '50; Hardy, '52; Moretti and Mescon, '56; Kopf, '57; BraunFalco, '58; Moretti et al., '60). There are, however, a few studies on phosphatases in embryos other than rat (Moog, '44; Johnson and Bevelander, '46).The present study deals with the distribution and activity of alkaline, acid, poly-and meta-phosphatases in the rat skin, and, further, a chronological followup of these enzymes from embryonic to adult stage.
MATERIALS AND METHODSAnimals. Nine embryos ( 3 embryos each at the seventh, fourteenth, and eighteen day of gestation), 2 new-born rats three hours after birth, 1 rat 10 days old, 1 rat 15 days old, and 5 adult rats of the Wistar strain, weighing approximately 100 g, were used in the present investigation.Rats were killed by a blow on the neck without anesthesia. Hair was cut. Skin specimens taken from the back were cut into small square pieces and immediately fixed in ice-cold 10% neutral formalin for 30 minutes. In the case of embryos, skin was removed from the back while they were still alive.Frozen sections, approximately 20 micra in thickness, were made and used for various enzymatic studies.Histochemical demonstrution of alhaline phosphatases (ALP.). Gomori's modified calcium cobalt method (Gomori, '52) was applied. The sections were incubated in the substrate medium at 37°C for 2 to 21/2 hours.The effect of various pHs (8.2, 9.2, and 10.0) on the activity of alkaline phosphatases was tested with 2% sodium P-glycerophosphate as substrate as well as with several other substrates, such as 0.2% glucose-1-phosphate (G-1-P), 0.2% glucose-6-phosphate (G-6-P) and 0.2% fructose-6-phosphate (F-6-P), using adult rats as well as embryos of the fourteenth day of gestation.
Histochemical demonstration of acid phosphatase (AC.P.).Gomori's lead acetate method (Gomorim '50) was used to demonstrate non-specific acid phosphatases. Frozen sections were incubated in the substrate medium for 30 to 60 minutes at 37°C.The effect of various pHs (4.4, 5.2 and 6.0) on the enzyme activity was also observed using adult rats and embryos in their fourteenth day of gestation.Histochemical demonstration of tripolyphosphatase ( T P P ) and hexametaphosphatase ( H M P ) . Berg's method (Berg, '60) was first used with tripolyphosphate, Na5P,OIo, and sodium hexametaphosphate, ( NaPos)6, as substrates. Since, however, the results of staining were inconsistent with this method, the media were then prepared by the second method used originally by Berg ('55) for the demonstration of inorganic polyphosphatase to obtain more consistent results.As was done in the cases of alkaline and acid phosphatases, the effect of various pHs (3.2, 4.2, 4.4, 4.8 and 5.4) ...
