THE BIOLOGICAL REALITY OF THE INTERLACUNAR NETWORK IN THE EMBRYONIC, CARTILAGINOUS, SKELETON - A THIAZINE DYE ABSOLUTE ETHANOL LR WHITE RESIN PROTOCOL FOR VISUALIZING THE NETWORK WITH MINIMAL TISSUE SHRINKAGE

Third toe phalanges of chicks aged 8-13 days in ovo and 7-day post-nat al rat femoral growth plate were examined to determine whether the int erlacunar network (IN), a structure with no lipoprotein membrane compo nent or cytoplasmic organelles, is a genuine component of young growth cartilage, In chick phalanges dehydrated by 70% (v/v) ethanol and LR White resin, variable metachromatic staining of the interlacunar netwo rk by toluidine blue and red staining by picro-Sirius red indicate the presence of glycosaminoglycans and collagen, The network in phalanges dehydrated by 80% (v/v) ethanol appears little different; however, th e network is much less widely detectable in phalanges dehydrated by 90 % (v/v) ethanol and, after dehydration by absolute ethanol, is almost completely undetectable. In contrast, when the young cartilage is perm eated by a thiazine dye such as toluidine blue, using a solution of dy e in the aldehyde fixative, the network is widely detectable, followin g dehydration by absolute ethanol, both in chick phalanges and in rat growth plate. Comparison of projected areas shows that the extent to w hich whole chick feet are found to have shrunk, by the time that they are photographed under LR White resin, is determined principally by th e extent of dehydration, by 70% (v/v) or absolute ethanol; post-shrink age areas are 33% or 35% of areas measured in buffer for 70% (v/v) eth anol/LR White resin and 71% or 75% for absolute ethanol/LR White resin (the higher value in each is for the toluidine blue treatment). The n etwork is thus present in radically shrunk tissue,,but, significantly, is also fully represented in tissue shrunk by only a conventional mar gin and is therefore not produced as an artefact by exceptional tissue shrinkage as has been suggested.