Heat-induced retrieval of immunogold labeling for nucleobindin and osteoadherin from Lowicryl sections of bone.
The main purpose of this study was to examine whether antigens can be retrieved by heating Lowicryl sections of paraformaldehyde-fixed (PFF) tissues. Thus the intensity of the immunogold signal for two bone proteins (Nucleobindin (Nuc) and osteoadherin (OSAD)) was compared in retrieved and non-retrieved sections of PFF rat bone. As an additional experiment, the effect of antigen retrieval (for Nuc) in sections of tissue primary stabilized by high pressure freezing with subsequent freeze substitution (HPF-FS) was studied. Finally, the tissue distribution patterns of Nuc labeling were compared in non-retrieved HPF-FS sections to that of retrieved and non-retrieved PFF sections. Antigen retrieval in Lowicryl sections of PFF tissues showed significantly enhanced labeling intensity for both proteins in all compartments where they are known to occur. Retrieved PFF Lowicryl sections showed only minor ultrastructural differences compared to non-retrieved ones. Retrieval of HPF-FS sections exhibited no enhancement of labeling but rather a slight reduction, which was significant in the cytoplasm and in cartilage. Furthermore, striking ultrastructural differences were observed in retrieved HPF-FS sections compared to non-retrieved ones with loss of coherence and structure in sections subjected to heating. Comparison of the distribution patterns of Nuc in the sections of PFF and HPF-FS tissues showed discrepancy in most compartments. Antigen retrieval by heating Lowicryl sections of PFF tissues significantly enhances immunogold labeling in all cell compartments where the bone proteins are known to occur. However, the procedure may distort the tissue distribution pattern of bone proteins.
Solberg
Lene B
LB
Institute and Department of Pathology, University of Oslo, Rikshospitalet-Radiumhospitalet Trust, Oslo, Norway.
Melhus
Gunhild
G
Brorson
Sverre-Henning
SH
Wendel
Mikael
M
Reinholt
Finn P
FP
D000180:Acrylic Resins; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism* / Q000648:ultrastructure*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005557:Formaldehyde; D017110:Freeze Substitution; D006358:Hot Temperature; D007150:Immunohistochemistry / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011108:Polymers; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D016707:Tissue Fixation
D000180:Acrylic Resins; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011108:Polymers; D011509:Proteoglycans; C113360:osteoadherin; C050065:Lowicryl HM23; D005557:Formaldehyde; C003043:paraform