Combined use of malachite green fixation and PLA2-gold complex technique to localize phospholipids in areas of early calcification of rat epiphyseal cartilage and bone.
Two cytochemical methods (malachite green fixation and PLA2-gold complex technique) were combined in order to detect the presence and ultrastructural distribution of phospholipids in epiphyseal cartilage and metaphyseal bone of 25-day-old rats. Chondrocytes and osteoblasts showed a more intense PLA2-gold complex labeling of rough endoplasmic reticulum than mitochondria and plasma membranes. Roundish osmiophilic, electron-dense, lightly labeled structures were visible at the periphery of the cells. In areas of early mineralization of cartilage, not all the matrix vesicles were labeled by PLA2-gold complex. Calcification nodules showed intense labeling in comparison with the surrounding, lightly labeled uncalcified matrix; gold particles were chiefly found at their periphery. Calcification nodules of metaphyseal bone were deeply electron-dense after glutaraldehyde-malachite green fixation; PLA2-gold labeling was mainly found in connection with crystals, whereas the uncalcified matrix was weakly labeled. Calcified bone matrix showed a heavy labeling with randomly scattered gold particles. When pre-embedding decalcification was carried out in the presence of malachite green, a good preservation of organic component was obtained. The PLA2-gold positivity of decalcified areas of cartilage and bone confirmed the presence of phospholipids in mineralized matrix.
Silvestrini
G
G
Department of Experimental Medicine and Pathology, La Sapienza University, Rome, Italy.
Zini
N
N
Sabatelli
P
P
Mocetti
P
P
Maraldi
N M
NM
Bonucci
E
E
D000818:Animals; D002114:Calcinosis / Q000503:physiopathology; D004396:Coloring Agents / Q000737:chemistry*; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D004853:Epoxy Resins / Q000737:chemistry / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D007150:Immunohistochemistry; D010718:Phosphatidylserines / Q000378:metabolism*; D010741:Phospholipases A / Q000737:chemistry; D054467:Phospholipases A2; D051381:Rats; D017208:Rats, Wistar; D012394:Rosaniline Dyes / Q000737:chemistry*; D016707:Tissue Fixation
D004396:Coloring Agents; D004853:Epoxy Resins; D010718:Phosphatidylserines; D012394:Rosaniline Dyes; C005095:malachite green; C004875:EPON; D010741:Phospholipases A; D054467:Phospholipases A2; D005976:Glutaral