scholarly journals Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy.

1984 ◽  
Vol 32 (11) ◽  
pp. 1217-1223 ◽  
Author(s):  
L G Altman ◽  
B G Schneider ◽  
D S Papermaster
Keyword(s):  
Room Temperature ◽  
Dimethyl Formamide ◽  
Point Counting ◽  
Rabbit Antibody ◽  
Electron Microscope Level ◽  
Thin Sections ◽  
Kidney Microsomes ◽  
Embedding Medium ◽  
Lowicryl K4m ◽  
Accelerated Protocol

Lowicryl K4M (K4M) was recently introduced as an embedding medium for immunocytochemistry at the electron microscope level (BL Armbruster, E Carlemalm, R Chiovetti, RM Garavito, JA Hobot, E Kellenberger, W Villiger (1982):J Microsc 126:77 and E Carlemalm, M Garavito, W Villiger (1982):J Microsc 126:123). While earlier protocols of fixation and embedding required 4-6 days, the present method has reduced the processing time by accelerating both dehydration of tissues and polymerization of K4M so that tissues can be prepared for sectioning within 4 hr. The immunocytochemical labeling density was quantitated in order to determine relative antigen preservation in tissues embedded by the accelerated protocol as compared to slower K4M embedding techniques and to tissues embedded in glutaraldehyde-cross-linked bovine serum albumin (BSA). Thin sections of Bufo marinus kidney were labeled with rabbit antibody to Na+,K+ATPase alpha chain catalytic subunit isolated from B. marinus kidney microsomes (M Girardet, K Geering, JM Frantes, D Geser, BC Rossier, JP Kraehenbuhl, C Bron (1981):Biochemistry 20:6684). B. marinus retinas were labeled with rabbit anti-opsin. After fixation in paraformaldehyde(3%)-glutaraldehyde(3%), tissues were washed in buffer, dehydrated in 50, 75, and 90% dimethyl-formamide (DMF, 10 min each); K4M:DMF, 1:2 (15 min); K4M:DMF, 1:1, (20 min); K4M (25 min); K4M (30 min) at room temperature and transferred in fresh K4M to BEEM capsules for exposure to ultraviolet light (GE 15 watt, Black-lite, 10 cm, 45 min or less) at 4 degrees C. Thin sections were labeled successively with antibody, biotinylated sheep anti-rabbit F(ab')2 and avidin-ferritin. Ferritin labeling densities were determined by point counting. High labeling densities were observed with both antibodies, equaling or exceeding levels of labeling by slower protocols or embedment in BSA.

scholarly journals Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.

1982 ◽  
Vol 93 (1) ◽  
pp. 223-229 ◽  
Author(s):  
J Roth ◽  
E G Berger
Keyword(s):  
Golgi Apparatus ◽  
Hela Cells ◽  
Protein A ◽  
Rabbit Antibody ◽  
Concerted Action ◽  
Electron Microscope Level ◽  
Thin Sections ◽  
Thiamine Pyrophosphatase ◽  
In Trans ◽  
Lowicryl K4m

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.

HACH: A Polymer Designed to Optimize Protein Antigen Localization

1997 ◽  
Vol 3 (4) ◽  
pp. 321-331 ◽  
Author(s):  
J.B. Olesen ◽  
C.A. Heckman ◽  
A. Lukinius ◽  
D.W. Schwab ◽  
D.V. Upite ◽  
...  
Keyword(s):  
Room Temperature ◽  
Limit Of Detection ◽  
Protein Antigens ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Thin Sections ◽  
Antigen Localization ◽  
Lipoamide Dehydrogenase ◽  
Embedding Medium ◽  
Lowicryl K4m

Abstract: The purpose of this study was to determine whether a polymer could be formed from relatively innocuous monomeric ingredients and, if so, if it might serve as a suitable embedding medium for maximizing antigen retention. Such a polymer, HACH, was made up from a mixture of 2-hydroxyhexanedial and carbohydrazide. It polymerized spontaneously at room temperature within 24 hr. Preservation of protein antigenicity and subsequent immunocytochemical localization were demonstrated by three methods. To determine whether protein antigens were retained up to the polymerization stage, we studied hemoagglutination of red blood cells using antibodies directed against their protein antigens. In these trials, HACH-treated cells exhibited the same agglutination responses as control, untreated cells. Second, a guinea pig antibody was used to immunodecorate insulin in β cells of the islets of Langerhans. The number of gold particles, indicating sites where the antibody was bound, was several-fold greater in HACH- than in Lowicryl K4M-embedded pancreatic β cells. To assess the limit of detection of protein antigens in thin sections, an example of a protein present in mitochondria, lipoamide dehydrogenase, was also studied. An indirect procedure for immunodecoration, employing rabbit immunoglobulin G followed by gold-tagged secondary antibody, indicated that the enzyme was present at several sites within cross-sectioned mitochondria. The results suggest that the HACH polymer will be useful for the localization of antigens that are present in relatively few copies.

scholarly journals Lowicryl K4M embedding of brain tissue for immunogold electron microscopy.

1985 ◽  
Vol 33 (9) ◽  
pp. 969-973 ◽  
Author(s):  
K L Valentino ◽  
D A Crumrine ◽  
L F Reichardt
Keyword(s):  
Electron Microscopy ◽  
Brain Tissue ◽  
Room Temperature ◽  
Uv Light ◽  
Uranyl Acetate ◽  
Immunogold Electron Microscopy ◽  
Cacodylate Buffer ◽  
Perfusion Fixation ◽  
Embedding Medium ◽  
Lowicryl K4m

We present methods for embedding brain tissue in Lowicryl K4M embedding medium and localizing antigens using postembedding immunogold techniques. After perfusion fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, blocks of rat brain were placed in 2% aqueous uranyl acetate for 1 hour, dehydrated in 50%, 70%, and 95% ethanol, infiltrated with Lowicryl/ethanol mixtures (1:2 for 10 min, 1:1 for 15 min) and 100% Lowicryl (20 min and 25 min). Polymerization was carried out under UV light for 24-48 hours at room temperature. Several neural antigens, including three different synaptic vesicle proteins and an enzyme associated with the postsynaptic density, were localized by this technique, indicating that this procedure may have wide applicability.

scholarly journals Preparation of Geological and Biological TEM Specimens by Embedding in Sulfur

2004 ◽  
Vol 12 (5) ◽  
pp. 28-31 ◽  
Author(s):  
Richard C. Hugo ◽  
Sherry L. Cady
Keyword(s):  
Room Temperature ◽  
Vacuum Chamber ◽  
Ion Beam ◽  
Liquid Sulfur ◽  
Thin Sections ◽  
Geological Materials ◽  
Thin Sectioning ◽  
High Vapor ◽  
Embedding Medium ◽  
Suitable Technique

The technique of embedding TEM specimens in polymer resins for subsequent ultra-thin sectioning is well established. For geological materials, this protocol prevents specimen damage introduced by ion beam thinning, and is often used for preparing friable or porous materials. However, traditional polymer-based embedding resins may introduce organic carbon contaminants, producing artifacts in carbon analyses. Thus, a carbon-free substitute for traditional embedding media is required to ensure accurate carbon analyses of embedded and ultramicrotomed TEM specimens.A suitable technique that was first reported by Bradley is to embed specimens in pure sultur. In this technique, the specimen is immersed in liquid sulfur, the liquid is solidified, and the resulting block is ultramicrotomed as with traditional resins. Since sulfur has a high vapor pressure at room temperature, the ultra-thin sections are then placed in a separate vacuum chamber to sublimate the sulfur, yielding TEM specimens free of any embedding medium.

scholarly journals In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes.

1986 ◽  
Vol 102 (5) ◽  
pp. 1646-1653 ◽  
Author(s):  
M Binder ◽  
S Tourmente ◽  
J Roth ◽  
M Renaud ◽  
W J Gehring
Keyword(s):  
Electron Microscope ◽  
Protein A ◽  
Microscope Level ◽  
Gold Complex ◽  
Electron Microscope Level ◽  
Thin Sections ◽  
Hybrid Detection ◽  
Ultrathin Sections ◽  
Lowicryl K4m

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.

Immunoelectron microscopy of retinoblastoma

1987 ◽  
Vol 45 ◽  
pp. 926-927
Author(s):  
M.M. Rodrigues ◽  
J. Hackett ◽  
B. Wiggert ◽  
G. Chader
Keyword(s):  
Immunoelectron Microscopy ◽  
Room Temperature ◽  
Uv Light ◽  
Immunocytochemical Staining ◽  
Dimethyl Formamide ◽  
Thin Sections ◽  
Specific Proteins ◽  
Excised Tissues ◽  
Secondary Antibodies ◽  
20 Nm

The histogenesis of retinoblastoma is uncertain. The derivation of this tumor has been attributed to neuronal and/or glial cells, by routine transmission electron microscopy and light microscopic immunocytochemical staining. Immunoelectron microscopy can provide more precise localization of retinal proteins. We investigated the immunoelectron microscopic localization of photoreceptor cell specific proteins to determine their distribution in eyes enucleated for retinoblastoma.Surgically excised tissues were fixed in 1% glutaraldehyde 2%- paraformaldehyde in 0.1M phosphate buffer for 2 h at 4°C, dehydrated in 50%, 75% and 90% dimethyl-formamide and embedded in Lowicryl. Polymerization of Lowicryl was performed by exposure to UV light for 24 h at 4°, followed by polymerization at room temperature for 48-72 hr. Thin sections were incubated for 1 h at room temperature with primary antibodies against interphotoreceptor retinoid-binding protein (IRBP) (1:200 dilution), S-antigen (1:500 dilution) and opsin (1:500 dilution). Appropriate secondary antibodies either to mouse or rabbit IgG (1:20 dilution) were linked to colloidal gold (10-20 nm) and incubated for 30 min.

Current Applications of Lowicryl-K4M in Immunocytochemistry

1985 ◽  
Vol 43 ◽  
pp. 430-433
Author(s):  
Lawrence G. Altman ◽  
Barbara G. Schneider ◽  
David S. Papermaster
Keyword(s):  
Immunoelectron Microscopy ◽  
Polymerization Time ◽  
Morphometric Data ◽  
Thin Sections ◽  
Resin Infiltration ◽  
Entire Process ◽  
Antigenic Sites ◽  
Rapid Fixation ◽  
Embedding Medium ◽  
Lowicryl K4m

The introduction of Lowicryl-K4M as an embedding medium for immunoelectron microscopy has increased the number of options now available when attempting to localize antigenic sites on thin sections. Here we review some modifications of the manufacturer's original embedding protocol (5-6 days) by instituting changes that minimize antigen exposure to a number of potentially deleterious conditions while accelerating the entire process. Fixation and resin infiltration may be completed in three hours (room temp.) and block polymerization (0-4•) is often possible in less than 60 minutes. This rapid approach was tested for antigen preservation in Bufo marinus kidney (Na+,K+ ATPase alpha chain catalytic subunit) and retina (opsin) by comparing morphometric data (ferritin particles/μm2) against an alternate (intermediate) technique which employed the same rapid fixation/ dehydration/ Infiltration but with a more extended polymerization time of 2-3 days.

Cell Fine Structure as Revealed in Dessicator-Dried Resinless Sections

1981 ◽  
Vol 39 ◽  
pp. 622-623
Author(s):  
R. P. Becker ◽  
J. J. Wolosewick ◽  
J. Ross-Stanton
Keyword(s):  
Fine Structure ◽  
Tannic Acid ◽  
Three Dimensional ◽  
Albino Rats ◽  
Cell Organelles ◽  
Vascular Perfusion ◽  
Thin Sections ◽  
Carbon Coated ◽  
Embedding Medium ◽  
Polyethylene Glycol 6000

Methodology has been introduced recently which allows transmission and scanning electron microscopy of cell fine structure in semi-thin sections unencumbered by an embedding medium. Images obtained from these “resinless” sections show a three-dimensional lattice of microtrabeculfee contiguous with cytoskeletal structures and membrane-bounded cell organelles. Visualization of these structures, especially of the matiiDra-nous components, can be facilitated by employing tannic acid in the fixation step and dessicator drying, as reported here.Albino rats were fixed by vascular perfusion with 2% glutaraldehyde or 1.5% depolymerized paraformaldehyde plus 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4). Tissues were removed and minced in the fixative and stored overnight in fixative containing 4% tannic acid. The tissues were rinsed in buffer (0.2M cacodylate), exposed to 1% buffered osmium tetroxide, dehydrated in ethyl alcohol, and embedded in pure polyethylene glycol-6000 (PEG). Sections were cut on glass knives with a Sorvall MT-1 microtome and mounted onto poly-L-lysine, formvar-carbon coated grids while submerged in a solution of 95% ethanol containing 5% PEG.

Lattice imaging and pore structure of β ferric oxyhydroxide

1978 ◽  
Vol 36 (1) ◽  
pp. 264-265
Author(s):  
T. Baird ◽  
J.R. Fryer ◽  
S.T. Galbraith
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Bulk Material ◽  
Model Structure ◽  
Bulk Crystals ◽  
Lattice Imaging ◽  
Thin Sections ◽  
Ferric Oxyhydroxide ◽  
Resolution Electron ◽  
Hydrolysis Of

Introduction Previously we had suggested (l) that the striations observed in the pod shaped crystals of β FeOOH were an artefact of imaging in the electron microscope. Contrary to this adsorption measurements on bulk material had indicated the presence of some porosity and Gallagher (2) had proposed a model structure - based on the hollandite structure - showing the hollandite rods forming the sides of 30Å pores running the length of the crystal. Low resolution electron microscopy by Watson (3) on sectioned crystals embedded in methylmethacrylate had tended to support the existence of such pores.We have applied modern high resolution techniques to the bulk crystals and thin sections of them without confirming these earlier postulatesExperimental β FeOOH was prepared by room temperature hydrolysis of 0.01M solutions of FeCl3.6H2O, The precipitate was washed, dried in air, and embedded in Scandiplast resin. The sections were out on an LKB III Ultramicrotome to a thickness of about 500Å.

Immunogold localization of HMB-45 antigen in pulmonary lymphangiomyomatosis

1995 ◽  
Vol 53 ◽  
pp. 998-999
Author(s):  
J.M. Minda ◽  
E. Dessy ◽  
G. G. Pietra
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Muscle Cells ◽  
Ultrastructural Localization ◽  
Reproductive Age ◽  
Thin Sections ◽  
Smooth Muscle Proliferation ◽  
Hmb 45

Pulmonary lymphangiomyomatosis (PLAM) is a rare disease occurring exclusively in women of reproductive age. It involves the lungs, lymph nodes and lymphatic ducts. In the lungs, it is characterized by the proliferation of smooth muscle cells around lymphatics in the bronchovascular bundles, lobular septa and pleura The nature of smooth muscle proliferation in PLAM is still unclear. Recently, reactivity of the smooth muscle cells for HMB-45, a melanoma-related antigen has been reported by immunohistochemistry. The purpose of this study was the ultrastructural localization of HMB-45 immunoreactivity in these cells using gold-labeled antibodies.Lung tissue from three cases of PLAM, referred to our Institution for lung transplantation, was embedded in either Poly/Bed 812 post-fixed in 1% osmium tetroxide, or in LR White, without osmication. For the immunogold technique, thin sections were placed on Nickel grids and incubated with affinity purified, monoclonal anti-melanoma antibody HMB-45 (1:1) (Enzo Diag. Co) overnight at 4°C. After extensive washing with PBS, grids were treated with Goat-anti-mouse-IgG-Gold (5nm) (1:10) (Amersham Life Sci) for 1 hour, at room temperature.

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