Microwave fixation improves antigenicity of glutaraldehyde-sensitive antigens while preserving ultrastructural detail.
Microwave fixation for electron microscopy has been used primarily for post-embedding immunocytochemistry. The present study examined the ability of microwave fixation to preserve the antigenicity of glutaraldehyde-sensitive antigens for pre-embedding immunocytochemistry. Five monoclonal antibodies (MAbs) directed against cell surface components of rat mast cells were tested. The MAbs failed to show any labeling of conventionally fixed rat bone marrow-derived mast cells even at glutaraldehyde concentrations as low as 0.1%. Strong staining of mast cell plasma membranes was seen when bone marrow was initially fixed with 2% formaldehyde and then refixed in 2% glutaraldehyde/2% formaldehyde after immunostaining. However, the ultrastructural preservation of the cells was poor. Antigenicity and morphological detail were both preserved when bone marrow was fixed in 0.05% glutaraldehyde/2% formaldehyde for 4 sec in a 550-W microwave oven. With this method, mast cells in various stages of maturation as well as cells that did not contain granules were immunoreactive. This method should prove useful with antigens from many different cell types that are sensitive to glutaraldehyde fixation.
Jamur
M C
MC
Departamento de Biologia Celular, Universidade Federal do Parana, Curitiba, Pr, Brasil.
Faraco
C D
CD
Lunardi
L O
LO
Siraganian
R P
RP
Oliver
C
C
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D001854:Bone Marrow Cells; D002462:Cell Membrane / Q000648:ultrastructure; D005260:Female; D005557:Formaldehyde; D005976:Glutaral; D006652:Histological Techniques; D008297:Male; D008407:Mast Cells / Q000528:radiation effects / Q000648:ultrastructure*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D008872:Microwaves; D051381:Rats; D017208:Rats, Wistar; D014021:Tissue Preservation
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005557:Formaldehyde; D005976:Glutaral