28
2. TISSUE EXPOSURE
large quantities in exposed situations, are apt to discourage investigators
from attempting massive perfusions or flushing operations with animals
much larger than a rat. It is then that aldehyde perfusions become particularly valuable methods.
2.7. Hyaluronidase Pretreatment
Dense connective tissue presents a formidable barrier to the penetration of osmium tetroxide fixatives. This is true whether one is considering an organ capsule, an adventitial sheath, or simply an irregularly
arranged tissue such as dermis.
When such tissues are to be fixed, they first may be exposed to a brief
treatment with strong hyaluronidase which presumably acts by depolymerizing the mucopolysaccharides. There is good reason to believe that
subsequently the fixative penetrates much more effectively (Pallie and
Pease, 1958).
The method which has been developed consists in using a commercial
preparation of lyophilized hyaluronidase available in 150 unit ampules
(Wydase, manufactured by Wyeth Laboratories Incorporated, Philadelphia, Pennsylvania). This is dissolved in 2-10 ml of normal saline or
Ringer's solution. It is either topically applied in situ to a surface to be
fixed, or small pieces or thin sheets of tissue are submerged in the solution. Reaction is complete within 5 minutes, so that longer exposures
should not be employed.
Fixative follows this treatment without intervening washing of any
sort. Three precautions seem necessary. The enzyme is effective at room
or body temperature, but not when iced. The enzyme is quickly destroyed
by body fluids, so that the surface to be prepared must be flushed with
an excess of the solution. In particular, any oozing blood must be flushed
away. In the case of excised tissue, the first bath is changed quickly to a
second and even a third bath, and agitation is maintained. Salt solutions
should not be used for preliminary flushing or washing although oozing
blood or tissue fluid can be blotted away with absorbent cotton.
The initial investigation of the use of hyaluronidase was unable to
detect any deleterious effect upon the subsequent preservation of tissue.
The method has not had wide application as yet, however, and so should
2. TISSUE EXPOSURE
large quantities in exposed situations, are apt to discourage investigators
from attempting massive perfusions or flushing operations with animals
much larger than a rat. It is then that aldehyde perfusions become particularly valuable methods.
2.7. Hyaluronidase Pretreatment
Dense connective tissue presents a formidable barrier to the penetration of osmium tetroxide fixatives. This is true whether one is considering an organ capsule, an adventitial sheath, or simply an irregularly
arranged tissue such as dermis.
When such tissues are to be fixed, they first may be exposed to a brief
treatment with strong hyaluronidase which presumably acts by depolymerizing the mucopolysaccharides. There is good reason to believe that
subsequently the fixative penetrates much more effectively (Pallie and
Pease, 1958).
The method which has been developed consists in using a commercial
preparation of lyophilized hyaluronidase available in 150 unit ampules
(Wydase, manufactured by Wyeth Laboratories Incorporated, Philadelphia, Pennsylvania). This is dissolved in 2-10 ml of normal saline or
Ringer's solution. It is either topically applied in situ to a surface to be
fixed, or small pieces or thin sheets of tissue are submerged in the solution. Reaction is complete within 5 minutes, so that longer exposures
should not be employed.
Fixative follows this treatment without intervening washing of any
sort. Three precautions seem necessary. The enzyme is effective at room
or body temperature, but not when iced. The enzyme is quickly destroyed
by body fluids, so that the surface to be prepared must be flushed with
an excess of the solution. In particular, any oozing blood must be flushed
away. In the case of excised tissue, the first bath is changed quickly to a
second and even a third bath, and agitation is maintained. Salt solutions
should not be used for preliminary flushing or washing although oozing
blood or tissue fluid can be blotted away with absorbent cotton.
The initial investigation of the use of hyaluronidase was unable to
detect any deleterious effect upon the subsequent preservation of tissue.
The method has not had wide application as yet, however, and so should