Aldehyde fixation is not necessary for the formation of “dark” neurons

By routine EM preparative techniques, the tissues which, collectively, separate maternal and fetal bloods in the fully formed chorioallantoic placenta of the rat have been shown to consist of three chorionic layers, or trophoblast, and a layer of allantoic capillary endothelium [Fig. 1]. Relationships between these layers are best demonstrated by special techniques, viz., cacodylate-buffered aldehyde fixation, collidine-buffered osmium tetroxide postfixation, and en bloc staining with uranyl acetate. By using this method on placentas at term, the cells of the outermost chorionic layer (Trophoblast 1) appear to be attached to each other by means of maculae adherentes which sometimes occur in clusters [Fig. 2].

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Complex Vesicles, Microtubules, and Cytoplasmic Filaments in the Endothelial Cells of Normal Dog Aorta

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100603 ◽

1968 ◽

Vol 26 ◽

pp. 172-173

Author(s):

C. N. Sun ◽

J. J. Ghidoni

Keyword(s):

Electron Microscopy ◽

Endothelial Cells ◽

Cross Sections ◽

Functional Significance ◽

Tubular Structures ◽

Aldehyde Fixation ◽

Cytoplasmic Filaments

Endothelial cells in longitudinal and cross sections of aortas from 3 randomly selected “normal” mongrel dogs were studied by electron microscopy. Segments of aorta were distended with cold cacodylate buffered 5% glutaraldehyde for 10 minutes prior to being cut into small, well oriented tissue blocks. After an additional 1-1/2 hour period in glutaraldehyde, the tissue blocks were well rinsed in buffer and post-fixed in OsO4. After dehydration they were embedded in a mixture of Maraglas, D.E.R. 732, and DDSA.Aldehyde fixation preserves the filamentous and tubular structures (300 Å and less) for adequate demonstration and study. The functional significance of filaments and microtubules has been recently discussed by Buckley and Porter; the precise roles of these cytoplasmic components remains problematic. Endothelial cells in canine aortas contained an abundance of both types of structures.

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Solubility of cytoskeletal proteins in immunohistochemistry and the influence of fixation.

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.4.8450200 ◽

1993 ◽

Vol 41 (4) ◽

pp. 609-616 ◽

Cited By ~ 27

Author(s):

B M Riederer ◽

R Porchet ◽

R A Marugg ◽

L I Binder

Keyword(s):

Protein Solubility ◽

Immunohistochemical Localization ◽

Influential Factors ◽

Cytoskeletal Proteins ◽

Fixed Tissue ◽

Aldehyde Fixation ◽

Cat Brain ◽

Spectrin Cytoskeleton ◽

Cryostat Sections

For accurate and quantitative immunohistochemical localization of antigens it is crucial to know the solubility of tissue proteins and their degree of loss during processing. In this study we focused on the solubility of several cytoskeletal proteins in cat brain tissue at various ages and their loss during immunohistochemical procedures. We further examined whether fixation affected either solubility or immunocytochemical detectability of several cytoskeletal proteins. An assay was designed to measure the solubility of cytoskeletal proteins in cryostat sections. Quantity and quality of proteins lost or remaining in tissue were measured and analyzed by electrophoresis and immunoblots. Most microtubule proteins were found to be soluble in unfixed and alcohol fixed tissues. Furthermore, the microtubule proteins remaining in the tissue had a changed cellular distribution. In contrast, brain spectrin and all three neurofilament subunits were insoluble and remained in the tissue, allowing their immunocytochemical localization in alcohol-fixed tissue. Synapsin I, a protein associated with the spectrin cytoskeleton, was soluble, and aldehyde fixation is advised for its immunohistochemical localization. With aldehyde fixation, the immunoreactivity of some antibodies against neurofilament proteins was reduced in axons unveiling novel immunogenic sites in nuclei that may represent artifacts of fixation. In conclusion, protein solubility and the effects of fixation are influential factors in cytoskeletal immunohistochemistry, and should be considered before assessments for a quantitative distribution are made.

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Microwaves and Heat in Aldehyde Fixation: Model Experiments with Bovine Serum Albumin

Methods ◽

10.1006/meth.1998.0614 ◽

1998 ◽

Vol 15 (2) ◽

pp. 119-122 ◽

Cited By ~ 9

Author(s):

D. Hopwood

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Model Experiments ◽

Aldehyde Fixation

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Immunocytochemical localization of histamine in secretory granules of rat peritoneal mast cells with conventional or rapid microwave fixation and an ultrastructural post-embedding immunogold technique.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.9.1506663 ◽

1992 ◽

Vol 40 (9) ◽

pp. 1247-1256 ◽

Cited By ~ 21

Author(s):

G R Login ◽

S J Galli ◽

A M Dvorak

Keyword(s):

Electron Microscopy ◽

Mast Cells ◽

Guinea Pig ◽

Secretory Granules ◽

Fixation Method ◽

Structural Localization ◽

Thin Sections ◽

Aldehyde Fixation ◽

Peritoneal Mast Cells ◽

Rat Peritoneal Mast Cells

We used a post-embedding immunogold labeling approach to define the fine-structural localization of histamine in rat peritoneal mast cells that were fixed using either standard aldehyde fixation or a fast microwave-aldehyde fixation method. Specimens were processed routinely for electron microscopy. Thin sections were exposed first to guinea pig antihistamine antiserum and then to gold-conjugated goat IgG directed against guinea pig IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with non-immune sera did not show labeling of mast cells. Adsorption of antihistamine antiserum with purified histamine or histamine bound to agarose showed a significant reduction (p less than 0.005) in granule staining. We also confirmed that our isolation procedures yielded functionally competent mast cells which released histamine when stimulated with sheep anti-rat IgE antiserum or with compound 48/80. These studies define the conditions of fixation for electron microscopy that are appropriate for the localization of histamine in the granule matrix of rat peritoneal mast cells.

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