Reducing labeling time of fluorescent molecules in thick tissue sections

AbstractA cell’s phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT—Spatially PhotoActivatable Color Encoded Cell Address Tags—to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function.

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The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100118989 ◽

1985 ◽

Vol 43 ◽

pp. 426-429

Author(s):

George H. Herbener ◽

Antonio Nanci ◽

Moise Bendayan

Keyword(s):

Tissue Section ◽

Colloidal Gold ◽

Unit Area ◽

Protein A ◽

Extracellular Proteins ◽

Gold Complex ◽

Second Step ◽

Igg Antibodies ◽

Tissue Sections ◽

Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

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Discovery of intracellular 2,8 dihydroxyadenine crystals in bovine lymphatic and hepatic cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012881x ◽

1987 ◽

Vol 45 ◽

pp. 906-907

Author(s):

W. E. Rigsby ◽

D. M. Hinton ◽

V. J. Hurst ◽

P. C. McCaskey

Keyword(s):

Polarized Light ◽

Paraffin Sections ◽

Tissue Samples ◽

Whole Cells ◽

Tissue Sections ◽

Hepatic Cells ◽

Slaughter House ◽

Mammalian Tissues ◽

Carbon Coated ◽

Cellular Components

Crystalline intracellular inclusions are rarely seen in mammalian tissues and are often difficult to positively identify. Lymph node and liver tissue samples were obtained from two cows which had been rejected at the slaughter house due to the abnormal appearance of these organs in the animals. The samples were fixed in formaldehyde and some of the fixed material was embedded in paraffin. Examination of the paraffin sections with polarized light microscopy revealed the presence of numerous crystals in both hepatic and lymph tissue sections. Tissue sections were then deparaffinized in xylene, mounted, carbon coated, and examined in a Phillips 505T SEM equipped with a Tracor Northern X-ray Energy Dispersive Spectroscopy (EDS) system. Crystals were obscured by cellular components and membranes so that EDS spectra were only obtainable from whole cells. Tissue samples which had been fixed but not paraffin-embedded were dehydrated, embedded in Spurrs plastic, and sectioned.

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Ultrastructural aspects of olfactory signal transduction and its development

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016844x ◽

1994 ◽

Vol 52 ◽

pp. 142-143

Author(s):

Bert Ph. M. Menco

Keyword(s):

Signal Transduction ◽

Olfactory Receptor ◽

Receptor Cell ◽

Freeze Substitution ◽

Luminal Surface ◽

Tissue Sections ◽

Olfactory Receptor Cells ◽

Receptor Cells ◽

Olfactory Signal ◽

Odor Molecule

Vertebrate olfactory receptor cells are specialized neurons that have numerous long tapering cilia. The distal parts of these cilia line the interface between the external odorous environment and the luminal surface of the olfactory epithelium. The length and number of these cilia results in a large surface area that presumably increases the chance that an odor molecule will meet a receptor cell. Advanced methods of cryoprepration and immuno-gold labeling were particularly useful to preserve the delicate ultrastructural and immunocytochemical features of olfactory cilia required for localization of molecules involved in olfactory signal-transduction. We subjected olfactory tissues to freeze-substitution in acetone (unfixed tissues) or methanol (fixed tissues) followed by low temperature embedding in Lowicryl K11M for that purpose. Tissue sections were immunoreacted with several antibodies against proteins that are presumably important in olfactory signal-transduction.

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Flat embeddment of monolayer cells, tissue sections, and other cellular materials attached onto glass substrate

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161394 ◽

1990 ◽

Vol 48 (3) ◽

pp. 766-767

Author(s):

Jeffrey P. Chang ◽

Jaang J. Wang

Keyword(s):

Present Report ◽

Cellular Materials ◽

Cover Glass ◽

Tissue Sections ◽

Embedding Technique ◽

Exfoliated Cells ◽

Black Paper ◽

Cell Concentrations ◽

Flat Embedding

Flat embeddment of certain specimens for electron microscopy is necessary for three classes of biological materials: namely monolayer cells, tissue sections of paraffin or plastics, as well as cell concentrations, exfoliated cells, and cell smears. The present report concerns a flat-embedding technique which can be applied to all these three classes of materials and which is a modified and improved version of Chang's original methodology.Preparation of coverglasses and microslides. Chemically cleaned coverglasses, 11 × 22 mm or other sizes, are laid in rows on black paper. Ink-mark one coner for identifying the spray-side of the glass for growing cells. Lightly spray with Teflon monomer (Heddy/Contact Inductries, Paterson, NO 07524, U.S.A.) from a pressurized can. Bake the sprayed glasses at 500°F for 45 min on Cover-Glass Ceramic Racks (A. Thomas Co. Philadelphia), for Teflon to polymerize.Monolayer Cells. After sterilization, the Teflon-treated coverglasses, with cells attached, are treated or fixed in situ in Columbia staining dishes (A. Thomas Co., Philadelphia) for subsequent processing.

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A combined pseudoreplica-immunocytochemical technique for research and diagnostic virology

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162065 ◽

1990 ◽

Vol 48 (3) ◽

pp. 900-901

Author(s):

Blayne Fritz ◽

Stanley J. Naides ◽

Kenneth Moore

Keyword(s):

Phosphotungstic Acid ◽

Immunogold Labelling ◽

Uranyl Acetate ◽

Distilled Water ◽

Clinical Specimens ◽

Tissue Sections ◽

Specimen Retrieval ◽

Transmission Electron ◽

Viral Particles ◽

Diagnostic Virology

The pseudoreplica method of staining viral particles for visualization by transmission electron microscopy is a very popular technique. The ability to concentrate clinical specimens while semi-embedding viral particles makes it especially well suited for morphologic and diagnostic virology. Immunolabelling viral particles with colloidal gold is a technique frequently employed by both research and diagnostic virologists. We have characterized a procedure which provides the advantage of both by modifying and combining pseudoreplica staining and immunogold labelling.Modification of specimen retrieval and delay of staining allows us to utilize pseudoreplica processed specimens within our standard immunogold labelling protocol. In brief, we absorbed samples onto 2% agarose, added.25% Formvar and wicked dry. We then floated the Formvar-virus film onto double distilled water, added grids and retrieved with parafilm. The Formvar-virus specimens were then treated as thin tissue sections within our standard two stage immunolabelling protocol. Following completion of immunogold labelling; each grid was negatively stained with phosphotungstic acid or uranyl acetate contrast stains.

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Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161989 ◽

1990 ◽

Vol 48 (3) ◽

pp. 884-885

Author(s):

Seiji Shioda ◽

Yasumitsu Nakai ◽

Atsushi Ichikawa ◽

Hidehiko Ochiai ◽

Nobuko Naito

Keyword(s):

Osmium Tetroxide ◽

Freeze Substitution ◽

Ultrastructural Localization ◽

Wistar Rat ◽

Neurosecretory Cells ◽

Rapid Freezing ◽

Sodium Metaperiodate ◽

Tissue Sections ◽

Ultrathin Sections ◽

Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

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Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100148642 ◽

1993 ◽

Vol 51 ◽

pp. 562-563

Author(s):

Hakan Ancin

Keyword(s):

Laser Scanning ◽

Population Analysis ◽

Three Dimensional ◽

Large Data ◽

Laser Scanning Confocal Microscopy ◽

Tissue Slices ◽

Scanning Confocal Microscopy ◽

Tissue Sections ◽

Spatially Adaptive ◽

Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

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