scholarly journals Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold.

1987 ◽  
Vol 35 (4) ◽  
pp. 419-425 ◽  
Author(s):  
H Mar ◽  
T Tsukada ◽  
A M Gown ◽  
T N Wight ◽  
D G Baskin
Keyword(s):  
Colloidal Gold ◽  
High Specificity ◽  
Ultrastructural Localization ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Antigenic Sites ◽  
Embedding Technique ◽  
Positive Immunostaining ◽  
Arterial Smooth Muscle Cells ◽  
Electron Microscopic Localization

Ultrastructural localization of growth hormone in rat anterior pituitary and of muscle-specific actin in rabbit arterial smooth muscle cells was accomplished with a post-embedment procedure using colloidal gold. Plastic sections (2 microns) were mounted on slides, deplasticized, immunostained with immunoglobulin-colloidal gold particles, re-embedded in Epon, and sectioned for electron microscopy. This procedure enabled light and electron microscopic localization of these intracellular antigens on the same section. Positive immunostaining was demonstrated with this procedure with a muscle-specific actin antibody which previously failed to localize antigenic sites by EM. The procedure described yielded staining of high specificity, with minimal background and well-preserved ultrastructure. This re-embedding technique is useful in situations where problems with post-embedding EM immunostaining exist and where correlative LM and EM immunostaining is essential.

Artifact-free electron microscopic immunocytochemistry on rat brain tissue

1991 ◽  
Vol 49 ◽  
pp. 272-273
Author(s):  
Veronika Burmeister ◽  
N. Ludvig ◽  
P.C. Jobe
Keyword(s):  
Microscopic Examination ◽  
Free Electron ◽  
Electron Microscopic Examination ◽  
Ultrastructural Localization ◽  
Rabbit Serum ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Phosphate Buffered Saline ◽  
Rat Brain Tissue ◽  
Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

scholarly journals Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells.

1981 ◽  
Vol 29 (11) ◽  
pp. 1289-1301 ◽  
Author(s):  
M C Willingham ◽  
S S Yamada ◽  
P J Bechtel ◽  
A V Rutherford ◽  
I H Pastan
Keyword(s):  
Cell Function ◽  
Ground Substance ◽  
Nonmuscle Myosin ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Localization Method ◽  
Preferential Localization ◽  
Cytoplasmic Ground Substance ◽  
Fibroblastic Cells ◽  
Electron Microscopic Localization

Nonmuscle myosin in the cytoplasm of cultured fibroblastic cells has been localized using light and electron microscopic immunocytochemistry. Antibodies to purified fibroblast myosin were produced in goat and rabbit and purified by affinity chromatography. Light microscopic immunofluorescence localization showed patterns similar to those previously published. Electron microscopic localization using the ethyldimethyl aminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation-permeabilization procedure and the ferritin bridge localization method produced quantifiable localization in intracellular sites with well-preserved ultrastructural morphology. Myosin was found to be a major component of the cytosol. It was distributed diffusely with no preferential localization on membranous organelles. Myosin was found to be slightly concentrated on the surface of microfilament-containing structures, including the subplasmalemmal microfilament mat and stress fibers, occasionally with an interrupted periodicity. However, no myosin was found in surface ruffles or microvilli. Morphometric quantitation showed that the majority of the cell's myosin was in the cytosol. This location is compatible with myosin being a component of the microtrabecular lattice of the cytoplasmic ground substance. The concentration of myosin in association with microfilaments was only twice that of the cytosol. This interpretation must be somewhat tempered by the possibility that some myosin bound to tightly packed actin may be inaccessible. The significance of this distribution of myosin in cell function is discussed.

Ultrastructural localization of serotonin and polypeptide YY (PYY) in endocrine cells of the human rectum.

1986 ◽  
Vol 34 (6) ◽  
pp. 719-726 ◽  
Author(s):  
A I Lukinius ◽  
J L Ericsson ◽  
M K Lundqvist ◽  
E M Wilander
Keyword(s):  
Colloidal Gold ◽  
Endocrine Cells ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Enterochromaffin Cells ◽  
Cell Type ◽  
Low Concentration ◽  
Antigenic Sites ◽  
Human Rectum ◽  
Ultrathin Sections

This study was performed with the aim of ultrastructurally localizing serotonin and polypeptide YY (PYY) in the endocrine cells of the human rectum. Existing basic methods for immunolocalization of antigenic sites in ultrathin sections were tested and modified to allow reproducible results with distinct localization of marker (colloidal gold probes coupled either to IgG or protein A). Probes signifying presence of serotonin were distinctly localized over all heteromorphous granules in argentaffin cells and, in addition, over some of the more monomorphous, rounded granules in a second cell type whose granules all were covered by probes showing localization of the PYY antigen. The results suggest that serotonin in endocrine cells of the gut is not confined to the enterochromaffin type but may also be present in trace amounts in non-enterochromaffin endocrine cells storing peptide hormones. Since probes marking sites of PYY were deposited over some heteromorphous granules in enterochromaffin cells, the evidence obtained also suggests that PYY may occur in low concentration in these cells. The distribution of probes in the sections indicated that antigenic sites were confined to granules in the cells.

scholarly journals Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections.

1982 ◽  
Vol 30 (7) ◽  
pp. 691-696 ◽  
Author(s):  
J Roth
Keyword(s):  
Protein A ◽  
Second Step ◽  
Staphylococcal Protein A ◽  
Paraffin Sections ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Antigen Localization ◽  
Color Mixing ◽  
Electron Microscopic Localization

The protein A-gold (pAg) complex, a useful reagent for electron microscopic localization of antigens in thin sections, is tested for its suitability as second step reagent in light microscopic immunohistochemistry. In addition, the preparation of colloidal silver, its complex formation with staphylococcal protein A and the application of the protein A-silver complex for antigen localization in paraffin sections is reported. The antigens were visualized in a two-step technique with specific antisera in the first incubation step and pAg or pA-silver as a general second step reagent. The pAg complex gives a red coloration of antigenic sites, whereas the pA-silver stained yellow. The contrasting color provided by the two immunocolloids allowed localization of two antigens in the same section. No color mixing occurred, showing that removal of the antibodies of the first staining sequence is unnecessary. Staining is virtually permanent with the light microscopic immunocolloid method. It is concluded that pAg and pA-silver complexes are useful as general second step reagents for the localization of a variety of antigens in paraffin sections.

Peripheral structures of plastids and ultrastructural localization of acid phosphatase and succinic dehydrogenase in a variegated Betula pubescens mutant

1975 ◽  
Vol 53 (10) ◽  
pp. 1072-1077 ◽  
Author(s):  
Niina Valanne
Keyword(s):  
Cell Wall ◽  
Acid Phosphatase ◽  
Succinic Dehydrogenase ◽  
Leaf Tissue ◽  
Ultrastructural Localization ◽  
Betula Pubescens ◽  
High Light ◽  
Electron Microscopic ◽  
White Leaf ◽  
Electron Microscopic Localization

In white leaf tissue from a variegated Betula pubescens mutant, acid-phosphatase activity is seen only in the vacuoles and the cell wall and no precipitation is evident inside the mutant plastids. The electron-microscopic localization of succinic dehydrogenase reveals activity in the intracristal spaces of mitochondria and the envelopes of both mitochondria and plastids. Since precipitation is not apparent inside the protrusions of the plastids and only the membrane occasionally shows a typical mitochondrial reaction, evidence seems to be lacking which indicates that mitochondria originate from plastids. The mitochondrion-like particles budding from the mutant plastids are considered to be proplastids. Peripheral vesicular structures are also seen in the chloroplasts of green leaf tissue from plants growing at high light intensities.

scholarly journals Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody.

1986 ◽  
Vol 34 (10) ◽  
pp. 1337-1342 ◽  
Author(s):  
K Bienz ◽  
D Egger ◽  
L Pasamontes
Keyword(s):  
Colloidal Gold ◽  
Physical Development ◽  
Primary Antibody ◽  
Silver Enhancement ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Double Labeling ◽  
Gold Marker

Electron microscopic sections, immunocytochemically labeled with colloidal gold, can be prepared for double labeling by applying the "EM-silver enhancement" procedure. This method, a photographic, so-called physical, development, increases the size of the gold marker to a predeterminable value and thereby inactivates the anti-species antibody present on the gold grain, thus allowing the labeling of a second antigen with antibody raised in the same species.

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