scholarly journals Use of tissue-free glycol methacrylate sections as semi-permeable membranes: a simple way to shorten incubation times and to improve localization in enzyme histochemistry.

1989 ◽  
Vol 37 (2) ◽  
pp. 173-176 ◽  
Author(s):  
P O Gerrits ◽  
R W Horobin ◽  
M J Hardonk
Keyword(s):  
Alkaline Phosphatase ◽  
Azo Dye ◽  
Enzyme Histochemistry ◽  
Diazonium Salt ◽  
Histochemical Staining ◽  
Semipermeable Membranes ◽  
Large Molecules ◽  
Glycol Methacrylate ◽  
Tissue Sections ◽  
Antibody Staining

Placing 2-microns sections of tissue-free glycol methacrylate on top of tissue sections is a simple way of forming semipermeable membranes to enhance enzyme histochemical staining. For demonstrating alkaline phosphatase in glycol methacrylate-embedded kidney by a standard azo dye method, such membranes enabled incubation times to be reduced to 1-2 hr, with azo dye reaction product being more crisply localized as compared to sections stained without membranes. Such effects are possible because the membranes are highly permeable to small molecules (e.g., substrate and diazonium salt), slightly permeable to molecules of moderate size (e.g., the final reaction product), and impermeable to large molecules (e.g., alkaline phosphatase and other tissue biopolymers). The implications of these findings for enzyme histochemistry and for enzyme-labeled antibody staining are discussed.

scholarly journals A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

1990 ◽  
Vol 38 (3) ◽  
pp. 437-442 ◽  
Author(s):  
C A Ziomek ◽  
M L Lepire ◽  
I Torres
Keyword(s):  
Alkaline Phosphatase ◽  
Azo Dye ◽  
Diazonium Salt ◽  
Primordial Germ Cells ◽  
Cell Types ◽  
Visual Observation ◽  
Staining Reaction ◽  
Intestinal Epithelial ◽  
Specificity And Sensitivity ◽  
F9 Teratocarcinoma

We describe a fluorescent histochemical technique for detection of nonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.

scholarly journals Enzyme histochemistry on freeze-substituted glycol methacrylate-embedded tissue.

1990 ◽  
Vol 38 (1) ◽  
pp. 95-101 ◽  
Author(s):  
G I Murray ◽  
S W Ewen
Keyword(s):  
Low Temperature ◽  
Enzyme Histochemistry ◽  
Aqueous Media ◽  
Freeze Substitution ◽  
Histochemical Demonstration ◽  
Enzyme Localization ◽  
Glycol Methacrylate ◽  
Tissue Sections ◽  
Wide Range ◽  
Tissue Specimens

We developed a method for histochemical demonstration of a wide range of enzymes in freeze-substituted glycol methacrylate-embedded tissue. Tissue specimens were freeze-substituted in acetone and then embedded at low temperature in glycol methacrylate resin. All enzymes studied (oxidoreductases, hydrolases) were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-substitution combined with low-temperature glycol methacrylate embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, maintenance of enzyme activity, and excellent tissue morphology.

scholarly journals Enzyme histochemistry on freeze-dried, resin-embedded tissue.

1989 ◽  
Vol 37 (5) ◽  
pp. 643-652 ◽  
Author(s):  
G I Murray ◽  
M D Burke ◽  
S W Ewen
Keyword(s):  
Enzyme Activity ◽  
Low Temperature ◽  
Enzyme Histochemistry ◽  
Aqueous Media ◽  
Histochemical Demonstration ◽  
Glycol Methacrylate ◽  
Tissue Sections ◽  
Freeze Dried ◽  
Wide Range ◽  
Tissue Specimens

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.

scholarly journals INFLUENCE OF CHEMICAL STRUCTURE ON THE RATE OF AZO COUPLING AND ITS SIGNIFICANCE IN HISTOCHEMICAL METHODOLOGY

1959 ◽  
Vol 7 (1) ◽  
pp. 50-65 ◽  
Author(s):  
MARVIN M. NACHLAS ◽  
THEODORE P. GOLDSTEIN ◽  
DAVID H. ROSENBLATT ◽  
MARVIN KIRSCH ◽  
ARNOLD M. SELIGMAN
Keyword(s):  
Aromatic Amines ◽  
Diazonium Salt ◽  
Tissue Sections ◽  
Coupling Techniques ◽  
Diazonium Ions ◽  
Coupling Rate ◽  
Diazonium Ion ◽  
Hydroxy Compounds ◽  
Positive Groups ◽  
Electro Negativity

Reliability of enzymatic localization in tissue sections by the simultaneous coupling techniques is dependent to a great extent upon the speed of coupling. Therefore, the influence on coupling rate of the structure of the diazonium ion and of the coupling component was studied. Electro-negative groups in the diazonium ion increase the rate of coupling, while the same groups in the coupling component decrease the rate. Electro-positive groups in the coupling component accelerate coupling, but slow it when present in the diazonium ion. The relation of coupling rate and electro-negativity of the substituents in the diazonium ion follows Hammett's equation (8). Although the relations is linear on coupling with aromatic hydroxy compounds, it is not so with aromatic amines. The most active diazonium ions showed no increase in coupling rate with aromatic amines. This suggests that for those enzymes hydrolyzing an ester link, increase of coupling rate might be accomplished by modifying the structure of either the coupling component used in the substrate or the diazonium salt. However, for enzymes splitting amide linkages, the only possibility of improving the localization is by modifying the structure of the coupling component.

Localized regions of egg cytoplasm that promote expression of endoderm-specific alkaline phosphatase in embryos of the ascidian Halocynthia roretzi

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 1-7 ◽  
Author(s):  
H. Nishida
Keyword(s):  
Alkaline Phosphatase ◽  
Histochemical Staining ◽  
Halocynthia Roretzi ◽  
Endoderm Differentiation ◽  
Initial Event ◽  
Cytoplasmic Factors ◽  
Early Embryos ◽  
Vegetal Pole ◽  
Cytoplasmic Determinants ◽  
Cytoplasmic Transfer

Embryogenesis in ascidians is known to be of the mosaic type, a property that suggests the presence of cytoplasmic factors in the egg which are responsible for specification of the developmental fates of early blastomeres. Endoderm cells are present in the trunk region of tadpole larvae, and these cells specifically express alkaline phosphatase (AP). Endoderm cells originate exclusively from blastomeres of the vegetal hemisphere of early embryos. To obtain direct evidence for cytoplasmic determinants of endoderm specification, we carried out cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various regions. Initially, presumptive-epidermis blastomeres (blastomeres from the animal hemisphere) were fused to cytoplasmic fragments from various regions of blastomeres of 8-cell embryos of Halocynthia roretzi, and development of endoderm cells was monitored by histochemical staining for AP. AP activity was observed only when presumptive-epidermis blastomeres were fused with cytoplasmic fragments from the presumptive-endoderm blastomeres. The results suggest that cytoplasmic factors that promote the initial event of endoderm differentiation (endoderm determinants) are present in endoderm-lineage blastomeres. Next, to examine the presence and localization of endoderm determinants in the egg, cytoplasmic fragments from various regions of unfertilized and fertilized eggs were fused with the presumptive-epidermis blastomeres. The results suggest that endoderm determinants are already present in unfertilized eggs, and that they are segregated by movements of the ooplasm after fertilization. Initially, these determinants move to the vegetal pole of the egg. Then, prior to the first cleavage, their distribution extends in the equatorial direction, namely, to the entire vegetal hemisphere from which future endoderm-lineage blastomeres are formed.

scholarly journals Placental Lactogen-I (PL-I) Target Tissues Identified with an Alkaline Phosphatase-PL-I Fusion Protein

1998 ◽  
Vol 46 (6) ◽  
pp. 737-743 ◽  
Author(s):  
Heiner Müller ◽  
Guoli Dai ◽  
Michael J. Soares
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Colorimetric Assay ◽  
Biological Activities ◽  
Placental Lactogen ◽  
Kidney Cells ◽  
Fetal Kidney ◽  
Transfected Cells ◽  
Tissue Sections ◽  
Labeling System

The rat placenta expresses a family of genes related to prolactin (PRL). Target tissues and physiological roles for many members of the PRL family have yet to be determined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I (PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat PL-I (AP-PL-I). The AP-PL-I construct was stably expressed in 293 human fetal kidney cells, as was the unmodified AP vector that served as a control. AP activity was monitored with a colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and PRL-like biological activities of the AP-PL-I fusion protein were demonstrated by immunoblotting and the Nb2 lymphoma cell proliferation assay, respectively. AP-PL-I specifically bound to tissue sections known to express the PRL receptor, including the ovary, liver, and choroid plexus. Binding of AP-PL-I to tissues was specific and could be competed with ovine PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and suggest that this labeling system may also be useful for monitoring the actions of other members of the PRL family.

Determination of Unconjugated and Total N-Acetyl-p-aminophenol (NAPA) in Urine and Serum

1967 ◽  
Vol 13 (3) ◽  
pp. 215-219 ◽  
Author(s):  
Karel P M Heirwegh ◽  
Johan Fevery
Keyword(s):  
Liver Disease ◽  
Acid Hydrolysis ◽  
Azo Dye ◽  
Acidic Medium ◽  
Diazonium Salt ◽  
Accurate Method ◽  
Total N ◽  
Hydrolysis Of ◽  
Urine And Serum

Abstract A sensitive and accurate method is described for the determination of N-acetyl-p-aminophenol (NAPA) and its metabolites in urine and serum. In strongly acidic medium, p-aminophenol (PAP) resulting from differential extraction and acid hydrolysis of total NAPA and unconjugated NAPA, is diazotized and the diazonium salt coupled with N-(1-naphthyl)ethylenediamine (NED) in the presence of ethanol. The blue azo dye formed is determined spectrophotometrically. Application to liver disease is briefly reported.

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