WormAtlas Anatomical Methods - Metal Mirror (Slam Freeze) Fixation (MMF)

2002 ◽  
Author(s):  
David H. Hall
Keyword(s):  
Author(s):  
Marie-Thérèse Nicolas

An alternative to aqueous chemical fixation consists in immobilizing physically the specimen by freezing it as fast as possible without using any cryoprotectant. This Fast Freeze Fixation (FFF) followed by Freeze Substitution (FS) avoids osmotic artefacts due to the slow penetration of liquid chemical fixative. Associated with Immuno-Gold labeling (IGS), FFF-FS allows a more precise localization of antigens.Using the bioluminescent bacteria Vibrio harveyi, a comparison of IGS with an antibody directed against its luciferase (enzyme of the luminescent reaction) has been done after liquid chemical fixation versus FFFFS. This later technique, beside an expected improvement of the ultrastructure always shows a better preservation of antigenicity and a lower background. In the case of FFF-FS technique (Figure 3):–labeling in acrylic resin (LRWhite) is 2 to 4 fold more intense than in epoxy resin (Epon),–but the ultrastructure is always better in Epon.–but the ultrastructure is always better in Epon.–The addition of fixatives in the substitution medium, results in a decrease of labeling which is more important in the case of a mixture of fixatives than with osmium tetroxide alone; with one exception: the substitution with glutaraldehyde which produces a dramatic increase in the density of the labeling but also, at the same time, a swelling of the cells of about 30%.


Cryobiology ◽  
1972 ◽  
Vol 9 (4) ◽  
pp. 322
Author(s):  
A.P. MacKenzie ◽  
T.A. Kuster ◽  
B.J. Luyet

1992 ◽  
Vol 102 (3) ◽  
pp. 475-485 ◽  
Author(s):  
MARIE-ODILE SOYER-GOBILLARD ◽  
MARIE-LINE GERAUD

Light-microscopy observation of the dinoflagellate Prorocentrum micans after silver-staining of the argyrophilic proteins of the nucleolar organizing region (Ag-NOR staining) showed the presence of nucleolar material throughout the vegetative cell cycle, and in particular during all the mitotic stages. This contrasts with the case in most higher eukaryotes, in which nucleoli disappear at the end of prophase and are reconstituted in daughter cells during telophase. Electron-microscope (EM) observations after conventional or fast-freeze fixation revealed that during interphase several functional nucleoli with three compartments (NORs, the fibrillogranular and the preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early prophase, when chromosomes are beginning to split, the nucleoli remain functional, whereas in late prophase they contain only a NOR and the granular component, and the chromosomes are surrounded by many protein masses. In early telophase, the nucleolar material coating the chromosomes migrates along with the chromosomes. Nucleologenesis occurs through the formation of prenucleolar bodies around lateral or telomeric nucleofilaments extruding from the chromosomes. Several chromosomes can contribute to the formation of one nucleolus. The behaviour of these ‘persistent nucleoli’ in a closed-nucleus model such as that of the dinoflagellates is discussed with regard to the higher eukaryotes.


1996 ◽  
Vol 70 (1) ◽  
pp. 85-89 ◽  
Author(s):  
C.J. Veltkamp ◽  
G.R. Richards ◽  
J.C. Chubb

AbstractFreeze fixation-dehydration was used for the first time in the preparation of attached Gyrodactylus for SEM viewing. The technique provided instant immobilization of specimens before death and negligible shrinkage throughout the fixation-dehydration process. Comparisons of sample means of two linear measurements of attached opisthaptors showed 20% more shrinkage of Gyrodactylus fixed using 10% neutral buffered formalin than those which were freeze fixed. Freeze fixation-dehydration was excellent for the study of gross external morphology of attached Gyrodactylus. However, the freeze fixation-dehydration process may cause disruption of intracellular structural components making delicate tissues brittle and more prone to damage during subsequent manipulation.


1982 ◽  
Vol 30 (2) ◽  
pp. 129-138 ◽  
Author(s):  
R W Dudek ◽  
G V Childs ◽  
A F Boyne

Quick-freeze fixation and freeze-dry methods were used successfully to obtain ultrastructural localization of insulin in the pancreatic beta cell by the unlabeled antibody-enzyme technique. In unosmicated freeze-fixed and freeze-dried islets, insulin was specifically demonstrated over the dense core of the secretory granules. In osmicated freeze-fixed and freeze-dried islets, insulin antigenicity withstood the osmium tetroxide vapor treatment. In addition, the surrounding ultrastructural resolution of morphologic features was significantly improved, which allowed insulin to be localized not only in secretory granules, but also in intracellular membranous compartments, with a degree of confidence not heretofore possible. Extracellular sites of insulin positivity in the islet were also localized and possible exocytotic activity for showing insulin release was observed.


PROTOPLASMA ◽  
1991 ◽  
Vol 160 (2-3) ◽  
pp. 159-166 ◽  
Author(s):  
Marie -Th�r�se Nicolas ◽  
D. Morse ◽  
Jean -Marie Bassot ◽  
J. Woodland Hastings

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