scholarly journals Immunostaining of keratin and vimentin in epidermis: comparison of different post-embedding immunogold techniques for electron microscopy.

1989 ◽  
Vol 37 (6) ◽  
pp. 863-868 ◽  
Author(s):  
G Mahrle ◽  
H J Schulze ◽  
A Kuhn ◽  
A Wevers
Keyword(s):  
Low Temperature ◽  
Polyclonal Antibodies ◽  
Cluster Formation ◽  
Linear Distribution ◽  
Immunogold Staining ◽  
Fixed Material ◽  
Staining Methods ◽  
Linear Alignment ◽  
Structural Preservation ◽  
Lowicryl K4m

The present study compares different post-embedding staining methods, including conventional and low-temperature embedding techniques, for demonstration of the keratin and vimentin cytoskeleton of epidermal cells, applying commercially available polyclonal and monoclonal antibodies. Immunogold staining (5-nm particles) was performed on the following material: (a) osmium-fixed and Durcupan-embedded material, etched with various solutions; (b) aldehyde-fixed material embedded in Lowicryl K4M at 4 degrees C and -35 degrees C; (c) aldehyde-fixed material embedded in Lowicryl K11M at -60 degrees C with and without cryoprotection with glycerol. In conventionally embedded material we failed to demonstrate intermediate filaments, whereas they were stained after low-temperature embedding with Lowicryl, i.e., K4M 4 degrees C, K4M -35 degrees C, and K11M -60 degrees C. The keratin and vimentin cytoskeleton reacted exclusively with polyclonal antibodies. The best results for antigenicity as well as structural preservation were achieved by post-embedding staining of K4M -35 degrees C-embedded material. Antibodies to keratin stained the cytoskeleton in keratinocytes of all epidermal layers. Filaments were decorated in a linear alignment. Antibodies to vimentin stained the cytoskeleton of Langerhans cells and melanocytes. In these cells a linear distribution pattern of the reaction product along the filaments and an extrafilamentous cluster formation were observed, indicating staining of vimentin and a vimentin-associated protein.

scholarly journals Laminin γ3 Chain Binds to Nidogen and Is Located in Murine Basement Membranes

2005 ◽  
Vol 280 (23) ◽  
pp. 22146-22153 ◽  
Author(s):  
Nikolaus Gersdorff ◽  
Eddie Kohfeldt ◽  
Takako Sasaki ◽  
Rupert Timpl ◽  
Nicolai Miosge
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Basement Membranes ◽  
Electron Microscopic ◽  
Immunogold Staining ◽  
Adult Mice ◽  
Epidermal Growth

Recently a novel laminin γ3 chain was identified in mouse and human and shown to have the same modular structure as the laminin γ1 chain. We expressed two fragments of the γ3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3–5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The γ3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the β2 VI/V fragment. The γ3 III3–5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the γ1 III3–5 fragment. These data suggested that laminins containing the γ3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against γ3 VI/V and γ3 III3–5 showed no cross-reaction with homologous fragments from the γ1 and γ2 chains of laminin and allowed the establishment of γ chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20–100-fold lower content of the γ3 chain compared with the γ1 chain in various tissue extracts of adult mice. The expression of γ3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the γ3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the γ3 chain to basement membranes of these tissues.

scholarly journals Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1503-1513 ◽  
Author(s):  
P Hourdille ◽  
E Heilmann ◽  
R Combrie ◽  
J Winckler ◽  
KJ Clemetson ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Membrane Systems ◽  
Immunogold Staining ◽  
Thin Sections ◽  
Surface Areas ◽  
Lowicryl K4m

Abstract Previous studies have shown a decreased binding of monoclonal antibodies (MoAbs) to glycoprotein (GP) Ib-IX complexes on thrombin- stimulated platelets, but the reason for this is poorly understood. We have used (1) immunofluorescence procedures and flow cytometry, and (2) immunogold staining and electron microscopy to investigate this phenomenon. Washed platelets were incubated with alpha-thrombin, adenosine diphosphate, or ionophore A23187 for increasing lengths of time. For alpha-thrombin, but not the other agonists, flow cytometry confirmed a dose- and time-dependent decrease in the binding of MoAbs specific for GP Ib alpha (AP-1, Bx-1), GP IX (FMC 25), or to the complex itself (SZ 1). Immunoglold staining performed using standard transmission or scanning electron microscopy high-lighted surface areas devoid of bound antibody. However, a quantitatively normal immunofluorescence was restored if paraformaldehyde-fixed, thrombin- stimulated platelets were permeabilized with Triton X-100 (Sigma Chemical Co, St Louis, MO) before MoAb addition, while immunogold staining was now seen to be concentrated within the interior of the platelet. Glutaraldehyde-fixed samples were then embedded in the resin Lowicryl K4M (Taab Laboratories Equipment Ltd, Aldermaston, England) and immunogold staining performed on thin sections using a polyclonal antibody to glycocalicin. An increased presence of GP Ib-IX complexes within surface-connected membrane systems of the thrombin-stimulated platelets was confirmed. Interestingly, GP Ib-IX movement was opposite to the thrombin-induced externalization of internal pools of GP IIb- IIIa complexes and of the alpha-granule membrane GP, GMP-140.

scholarly journals Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus.

1985 ◽  
Vol 101 (2) ◽  
pp. 488-499 ◽  
Author(s):  
M Vantard ◽  
A M Lambert ◽  
J De Mey ◽  
P Picquot ◽  
L J Van Eldik
Keyword(s):  
Animal Cell ◽  
Regulatory Mechanisms ◽  
Present Observation ◽  
Chromosome Movement ◽  
Animal Cells ◽  
Higher Plant ◽  
Mitotic Apparatus ◽  
Immunogold Staining ◽  
Staining Methods ◽  
Spindle Development

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

Study of cluster formation in low-temperature systems. Spectral manifestation of resonance dipole–dipole interactions between nondipole polyatomic molecules

2010 ◽  
Vol 36 (5) ◽  
pp. 439-447 ◽  
Author(s):  
A. N. Cherevatova ◽  
V. N. Bocharov ◽  
T. D. Kolomiitsova ◽  
D. N. Shchepkin ◽  
K. G. Tokhadze
Keyword(s):  
Low Temperature ◽  
Cluster Formation ◽  
Polyatomic Molecules ◽  
Spectral Manifestation ◽  
Dipole Interactions

scholarly journals STUDIES ON THE MICROTUBULES IN HELIOZOA

1969 ◽  
Vol 43 (1) ◽  
pp. 148-165 ◽  
Author(s):  
Lewis G. Tilney ◽  
Breck Byers
Keyword(s):  
Natural Selection ◽  
Low Temperature ◽  
Model Building ◽  
Stable Configuration ◽  
Equilibrium Conditions ◽  
Staining Methods ◽  
Selection Of

On the assumption that the double-coiled pattern of microtubules in the axoneme of Echinosphaerium might be due to links of two sizes between adjacent microtubules, we disassembled microtubules with low temperature and then carefully analyzed the patterns of microtubules that formed upon the addition of heat (22°C) or heat and D2O. Although most of the initial clusters of microtubules that formed could not be interpreted as part of an axoneme, the spacings between these microtubules were the same as that in the axoneme, 70 and 300 A. By model building we were able to show that all clusters that form, including stages in the formation of the axoneme and its 12-fold symmetry, could be explained by links of two sizes (70 and 300 A) and the substructure of the microtubule. We could demonstrate these links with improved staining methods. We suggest that nonaxonemal assemblies of microtubules may be eliminated by the natural selection of the most energetically stable configuration of microtubules, all others undergoing disassembly under equilibrium conditions. Model building further supports this suggestion since the model axoneme possesses more links per tubule than any other cluster found.

A simple device for low temperature polymerization of Lowicryl K4M resin

1985 ◽  
Vol 138 (1) ◽  
pp. 91-93 ◽  
Author(s):  
W. Völker ◽  
B. Frick ◽  
H. Robenek
Keyword(s):  
Low Temperature ◽  
Simple Device ◽  
Lowicryl K4m

scholarly journals Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

1991 ◽  
Vol 39 (6) ◽  
pp. 863-869 ◽  
Author(s):  
B Gee ◽  
M J Warhol ◽  
J Roth
Keyword(s):  
Horseradish Peroxidase ◽  
Polyclonal Antibodies ◽  
Light And Electron Microscopy ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Endogenous Peroxidase ◽  
Pre Treatment ◽  
Lowicryl K4m ◽  
Antigen Antibody

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

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