Novel labeling methods for em analysis of ultrathin cryosections

1996 ◽  
Vol 54 ◽  
pp. 894-895
Author(s):  
John M. Robinson ◽  
Toshihiro Takizawa
Keyword(s):  
Colloidal Gold ◽  
Model System ◽  
Human Neutrophils ◽  
Frozen Sections ◽  
Specific Granules ◽  
Gold Label ◽  
Ultrathin Cryosections ◽  
Ultrathin Frozen Sections ◽  
Particulate Probes ◽  
Enzyme Alkaline Phosphatase

Ultrathin cryosections are the most favorable material for localization of intracellular antigens with particulate probes (e.g., colloidal gold) in post-embedding immunocytochemistry. Cryosections are prepared under the most benign conditions as compared to embedding samples in various kinds of plastic media. Typically, higher labeling efficiencies can be achieved with ultrathin frozen sections than with plastic sections.We have utilized human neutrophils, the most abundant type of leukocyte, as a model system to explore labeling procedures for ultrathin cryosections. These labeling procedures can serve as alternative or complementary approaches to the traditional colloidal gold label. Neutrophils are characterized by the presence of numerous granules in their cytoplasm. For many years, the model for neutrophil structure has held that there are two granule types in these cells, the socalled azurophilic and specific granules. We have described an additional cytoplasmic compartment with unusual properties in neutrophils. This compartment is characterized by the presence of the enzyme alkaline phosphatase (Fig. 1).

scholarly journals Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections.

1994 ◽  
Vol 42 (12) ◽  
pp. 1615-1623 ◽  
Author(s):  
T Takizawa ◽  
J M Robinson
Keyword(s):  
Human Neutrophil ◽  
Colloidal Gold ◽  
Model System ◽  
Protein Markers ◽  
Silver Enhancement ◽  
Double Labeling ◽  
Cytoplasmic Granules ◽  
Gold Particles ◽  
Ultrastructural Immunocytochemistry ◽  
Specific Granules

We present a new application for the use of small immunogold particles (approximately 1.4-nm diameter) for ultrastructural immunocytochemistry. These small gold particles have been used on ultra-thin cryosections in conjunction with a silver enhancement procedure that does not degrade ultrastructural detail. We have used the human neutrophil as a model system, in which known protein markers of two different cytoplasmic granules were localized, in the development of this procedure. The 1.4-nm immunogold particles coupled with silver enhancement yield intense labeling for localization of lactoferrin, a marker for the specific granules, and myeloperoxidase, a marker for the azurophil granules. Double labeling in which one antigen was visualized with 1.4-nm gold and silver enhancement and a second antigen was detected with colloidal gold-IgG on the same ultra-thin cryosection was successfully achieved. We also show that 1.4-nm diameter immunogold particles penetrate into cryosectioned neutrophils to a greater extent than 5-nm or 10-nm immunogold probes. These results show that small immunogold particles, along with silver enhancement, are a useful addition to the immunolabeling methods available for use with ultra-thin cryosections.

scholarly journals Correlative Microscopy Using FluoroNanogold on Ultrathin Cryosections: Proof of Principle

1998 ◽  
Vol 46 (10) ◽  
pp. 1097-1102 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Kouki Suzuki ◽  
John M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Imaging Techniques ◽  
Human Neutrophils ◽  
Fluorescence Signal ◽  
Correlative Microscopy ◽  
Reporter System ◽  
Fluorescence And Electron Microscopy ◽  
Specific Granules ◽  
Ultrathin Cryosections ◽  
Proof Of Principle

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for highresolution correlation between immunofluorescence and electron microscopy.

Immunoferritin localization of intracellular antigens in positively stained frozen sections

1978 ◽  
Vol 36 (2) ◽  
pp. 168-169
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Surface Tension ◽  
Water Surface ◽  
Thin Layers ◽  
The Other ◽  
Positive Staining ◽  
Frozen Sections ◽  
The Past ◽  
Ultrathin Frozen Sections ◽  
Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

Localization of pancreatic enzymes in ultrathin frozen sections stained with metal labeled antibody

1978 ◽  
Vol 36 (2) ◽  
pp. 90-91
Author(s):  
Kenjiro Yasuda
Keyword(s):  
Fluorescent Antibody ◽  
Pancreatic Enzymes ◽  
Tissue Preparation ◽  
Zymogen Granules ◽  
Frozen Sections ◽  
En Bloc ◽  
Antibody Method ◽  
Ultrathin Frozen Sections ◽  
Fluorescent Antibody Method

Localization of amylase,chymotrypsinogen and trypsinogen in pancreas was demonstrated by Yasuda and Coons (1966), by using fluorescent antibody method. These enzymes were naturally found in the zymogen granules. Among them, amylase showed a diffuse localization around the nucleus, in addition to the zymogen granules. Using ferritin antibody method, scattered ferritin granules were also found around the Golgi area (Yasuda et al.,1967). The recent advance in the tissue preparation enables the antigen to be localized in the ultrathin frozen sections, by applying the labeled antibodies onto the sections instead of staining the tissue en bloc.The present study deals with the comparison of the localization of amylase and lipase demonstrated by applying the bismuth-labeled, peroxidase-labeled and ferritin-labeled antibody methods on the ultrathin frozen sections of pancreas, and on the blocks of the same tissue.

The Application of Ultracryotomy to Immunocytochemistry

1973 ◽  
Vol 31 ◽  
pp. 704-709
Author(s):  
R. G. Painter ◽  
K. T. Tokuyasu ◽  
S. J. Singer
Keyword(s):  
Frozen Sections ◽  
Protein Antigens ◽  
Carbon Coated ◽  
Antibody Conjugates ◽  
Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

Ultrathin frozen sections of yeast without aldehyde prefixation

1978 ◽  
Vol 36 (2) ◽  
pp. 58-59
Author(s):  
K. J. Böhm ◽  
a. E. Unger
Keyword(s):  
Aqueous Solutions ◽  
Biological Material ◽  
Yeast Cells ◽  
Frozen Sections ◽  
Good Preservation ◽  
Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

Immunogold localisation of endogenous immunoglobulin-G in ultrathin frozen sections of the human placenta

1989 ◽  
Vol 257 (3) ◽  
pp. 603-607 ◽  
Author(s):  
Lopa Leach ◽  
Bryan M. Eaton ◽  
J. Anthony Firth ◽  
Soli F. Contractor
Keyword(s):  
Human Placenta ◽  
Immunoglobulin G ◽  
Frozen Sections ◽  
Immunogold Localisation ◽  
Ultrathin Frozen Sections

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

scholarly journals Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules

1992 ◽  
Vol 284 (2) ◽  
pp. 513-520 ◽  
Author(s):  
S J Suchard ◽  
M J Burton ◽  
S J Stoehr
Keyword(s):  
Cytochalasin B ◽  
Anion Channel ◽  
Polymorphonuclear Leucocyte ◽  
Receptor Expression ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Surface Receptors ◽  
Specific Granules ◽  
Azurophil Granule ◽  
Disulphonic Acid

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

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