Rapid-freeze fixation and substitution improves tissue preservation of microspores and tapetum in Brassica napus

2000 ◽  
Vol 12 (4) ◽  
pp. 237-240 ◽  
Author(s):  
J. H. E. Ross ◽  
C. Milanesi ◽  
D. J. Murphy ◽  
Mauro Cresti
2009 ◽  
Vol 41 (6) ◽  
pp. 443-447 ◽  
Author(s):  
Claudio Milanesi ◽  
Rita Vignani ◽  
Andrea Ciacci ◽  
Alessandra Nardini ◽  
Marco Valenti ◽  
...  

PROTOPLASMA ◽  
1986 ◽  
Vol 131 (2) ◽  
pp. 153-165 ◽  
Author(s):  
Susan A. Lancelle ◽  
D. A. Callaham ◽  
P. K. Hepler

Author(s):  
J. G. Adams ◽  
M. M. Campbell ◽  
H. Thomas ◽  
J. J. Ghldonl

Since the introduction of epoxy resins as embedding material for electron microscopy, the list of new formulations and variations of widely accepted mixtures has grown rapidly. Described here is a resin system utilizing Maraglas 655, Dow D.E.R. 732, DDSA, and BDMA, which is a variation of the mixtures of Lockwood and Erlandson. In the development of the mixture, the Maraglas and the Dow resins were tested in 3 different volumetric proportions, 6:4, 7:3, and 8:2. Cutting qualities and characteristics of stability in the electron beam and image contrast were evaluated for these epoxy mixtures with anhydride (DDSA) to epoxy ratios of 0.4, 0.55, and 0.7. Each mixture was polymerized overnight at 60°C with 2% and 3% BDMA.Although the differences among the test resins were slight in terms of cutting ease, general tissue preservation, and stability in the beam, the 7:3 Maraglas to D.E.R. 732 ratio at an anhydride to epoxy ratio of 0.55 polymerized with 3% BDMA proved to be most consistent. The resulting plastic is relatively hard and somewhat brittle which necessitates trimming and facing the block slowly and cautiously to avoid chipping. Sections up to about 2 microns in thickness can be cut and stained with any of several light microscope stains and excellent quality light photomicrographs can be taken of such sections (Fig. 1).


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%.


Author(s):  
Richard Montione ◽  
Muhammad Ashraf

Osmolarity of a fixative vehicle has long been known to have an effect on the tissue preservation. An increase in tissue osmolarity occurs in ischemia-damaged tissue and affects the morphology. In this study, we examined cellular changes in ischemic rat myocardium induced by varying fixative toxicity.Rats were sacrificed by decapitation and the hearts immediately removed and retrogradily perfused through the aorta with anoxic Kurbs-Henseleit medium. Hearts were then placed in a bag with a small amount of medium at 37°C for 90 minutes. Hearts were perfusion-fixed using 2% glutaraldehyde in 0.1 M cacodylate buffer pH -7.3 at three osmolarities. The isotonic buffer was adjusted to 311 mOsm/kg using D-manitol. Hypertonic buffers were adjusted to 375 and 400 mOsm/kg. One-half hour after perfusion fixation, the hearts were sliced and cut into small blocks and allowed to fix overnight at 4°C. Blocks were post fixed in osmium, en bloc stained in uranyl acetate, dehydrated in ethanol and embedded in Spurr medium.


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