scholarly journals A NEW DIAZO REAGENT FOR SPECIFIC STAINING OF CONJUGATED BILIRUBIN IN TISSUE SECTIONS

1968 ◽  
Vol 16 (6) ◽  
pp. 419-427 ◽  
Author(s):  
V. J. DESMET ◽  
A.-M. BULLENS ◽  
J. DE GROOTE ◽  
K. P. M. HEIRWEGH
Keyword(s):  
Total Bilirubin ◽  
Diazonium Salt ◽  
Histochemical Demonstration ◽  
Specific Method ◽  
Bile Pigments ◽  
Specific Staining ◽  
Tissue Sections ◽  
A New Technique ◽  
Cryostat Sections ◽  
Conjugated Bilirubin

A new technique is presented for the specific histochemical demonstration of conjugated bilirubin in tissue sections, using the diazonium salt of ethylanthranilate. The specificity of this method was proved by using test materials and cholestatic tissue sections. There was no diffusion artifact under the prescribed working conditions. The method is applicable to fresh cryostat sections and frozen sections of cold, formol-calcium-fixed tissues. The method could not be satisfactorily applied for the staining of total bilirubin. The method using 2,4-dichloraniline remains the best available technique for the histochemical demonstration of total bilirubin, while the technique with ethylanthranilate proves to be the most specific method available for the demonstration of conjugated bile pigments.

scholarly journals Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.

1978 ◽  
Vol 26 (3) ◽  
pp. 157-162 ◽  
Author(s):  
A C Frasch ◽  
M E Itoiz ◽  
R L Cabrini
Keyword(s):  
Cytochrome Oxidase ◽  
Incubation Medium ◽  
Histochemical Demonstration ◽  
Tongue Muscle ◽  
Tissue Sections ◽  
Direct Light ◽  
Fixed Concentration ◽  
Set Up ◽  
Cryostat Sections

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.

scholarly journals HISTOCHEMICAL DEMONSTRATION OF MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE ACTIVITY IN TISSUE SECTIONS

1962 ◽  
Vol 10 (1) ◽  
pp. 65-74 ◽  
Author(s):  
MAX WACHSTEIN ◽  
MAIRE BRADSHAW ◽  
JOSÉ M. ORTIZ
Keyword(s):  
Adenosine Triphosphatase ◽  
Histochemical Demonstration ◽  
The Other ◽  
Mitochondrial Activity ◽  
Bile Canaliculi ◽  
Adenosine Triphosphatase Activity ◽  
Tissue Sections ◽  
Dog Kidney ◽  
Cryostat Sections ◽  
Neutral Formalin

A comparative study was made of the distribution of mitochondrial adenosine triphosphatase activity in several organs of rat and rabbit using both the calcium and lead techniques. Certain modifications in these techniques assured both improved intracellular morphology and considerable preservation of enzyme activity. Of particular importance was the low temperature (–2 to –3°C) of the neutral calcium-formol solution, and, following short fixation (lead method), a subsequent wash in neutral buffer. Mitochondrial activity was similar with both techniques, but the lead method proved to be less costly, less time-consuming, and above all, far less capricious than the calcium technique. In good preparations, the appearance of mitochondria is as clearly defined as in sections stained with non-enzymatic, conventional techniques. Long exposure of cryostat sections to formalin or preparation of sections from tissue blocks fixed in neutral formalin leads to the complete abolition of mitochondrial activity; on the other hand, it accentuates enzyme staining of other structures, such as, for instance, bile canaliculi in the liver and the secretory capillaries in the pancreas and salivary glands. It also visualizes the infolding membranes in certain tubules of the rat and dog kidney. It is assumed that formalin-fixation aids in the enzymato-morphologic distinction of these two different intracellular structures.

scholarly journals Immunohistochemical demonstration of H2 antigens in mouse tissue sections.

1983 ◽  
Vol 31 (7) ◽  
pp. 911-919 ◽  
Author(s):  
B A Ponder ◽  
M M Wilkinson ◽  
M Wood ◽  
J H Westwood
Keyword(s):  
Mouse Tissue ◽  
Double Staining ◽  
Specific Staining ◽  
Sandwich Technique ◽  
Tissue Sections ◽  
Immunohistochemical Demonstration ◽  
Mouse Chimeras ◽  
Mouse Tissues ◽  
Cellular Markers ◽  
Cryostat Sections

The immunohistological demonstration of H2 antigens in cryostat sections of a wide variety of mouse tissues is reported. The purpose in developing the method was to use H2 antigens as cellular markers in studies of mouse chimeras. Monoclonal anti-H2 antibodies were used, either with a hapten-sandwich technique using biotin or arsanilate, or as direct enzyme conjugates. The direct antibody-enzyme conjugates were simpler to use, provided an intensity of specific staining which was comparable to that obtained with the hapten-sandwich systems, and gave fewer problems of background when simultaneous double staining was attempted. The results provide a description of the distribution of H2 antigens in many of these tissues. The intensity of H2 staining varied widely from tissue to tissue, but also within tissues and between individual mice of the same litter. Quantitation by autoradiography suggests that there is a fivefold variation in available H2 antigen between tissues which are stained strongly or weakly by our technique.

Studies on the Determination of Bile Pigments

1964 ◽  
Vol 10 (5) ◽  
pp. 433-439 ◽  
Author(s):  
S L Jacobs ◽  
R J Henry ◽  
M Segalove
Keyword(s):  
Ethyl Acetate ◽  
Spectrophotometric Method ◽  
Total Bilirubin ◽  
Reference Standards ◽  
Fair Agreement ◽  
Bile Pigments ◽  
Acetone Precipitation ◽  
Discrepant Result ◽  
Conjugated Bilirubin

Abstract Tests for total bilirubin on 10 icteric sera by 5 diazo procedures and a spectrophotometric method showed fair agreement with most samples but an occasional grossly discrepant result. Results for free and conjugated bilirubin by diazo methods, acetone precipitation, and extraction of serum with ethyl acetate and chloroform were widely discrepant. Problems in documentation of accuracy of such procedures are discussed. It is concluded that unavailability of reference standards of bilirubin conjugates poses a serious obstacle to solution of the analytical problems.

Studies on the Determination of Bile Pigments

1964 ◽  
Vol 10 (2) ◽  
pp. 95-102 ◽  
Author(s):  
G W Stevenson ◽  
S L Jacobs ◽  
R J Henry
Keyword(s):  
Ethylene Glycol ◽  
Extraction Method ◽  
Total Bilirubin ◽  
Rapid Determination ◽  
Bile Pigments ◽  
Single Tube ◽  
Chloroform Layer ◽  
The Difference ◽  
Conjugated Bilirubin

Abstract The absorbance at 450 mµ of serum diluted with acidified ethylene glycol is used as a measure of the serum's total bilirubin content in a single-tube, single-extraction method for rapid determination of free and total bilirubin in 0.1 ml. of serum. With the addition of chloroform, free bilirubin is extracted into the chloroform but conjugated bilirubin remains in the ethylene glycol. Free bilirubin is determined from the absorbance at 450 mµ in the chloroform layer. Conjugated bilirubin is calculated from the difference. Absorbance readings at 520 mµ compensate for the absorbance at 450 mµ, due to hemoglobin in the serum. Comparison with the Malloy-Evelyn procedure showed equivalent levels of total but higher levels of conjugated bilirubin.

scholarly journals FLUORESCENCE METHODS FOR THE HISTOCHEMICAL DEMONSTRATION OF MONOAMINES. 3. SODIUM BOROHYDRIDE REDUCTION OF THE FLUORESCENT COMPOUNDS AS A SPECIFICITY TEST

1964 ◽  
Vol 12 (8) ◽  
pp. 582-586 ◽  
Author(s):  
HANS CORRODI ◽  
NILS-ÅKE HILLARP ◽  
GÖSTA JONSSON
Keyword(s):  
Sodium Borohydride ◽  
Histochemical Demonstration ◽  
Cellular Level ◽  
Specific Method ◽  
Tissue Sections ◽  
Specificity Test ◽  
Highly Sensitive ◽  
Formaldehyde Treatment ◽  
Sodium Borohydride Reduction ◽  
Fluorescent Compounds

It has been shown in previous papers that catecholamines and 5-hydroxytryptamine can under certain conditions be converted to highly fluorescent 6,7 - dihydroxy - 3,4 - dihydroisoquinolines and 6-hydroxy-3,4-dihydro-β-carboline respectively, and that this can be used as a highly sensitive and specific method for the histochemical demonstration of the monoamines at the cellular level. In the present paper it is shown that the fluorescent compounds are very readily reduced by sodium borohydride to the corresponding, non-fluorescent 1,2,3,4-tetrahydro-compounds—even if they are present in a non-extractable state in dried serum albumin spots or in tissue sections—and that the fluorescence can be regenerate by renewed formaldehyde treatment. The non-specific fluorescence ( e. g. autofluorescence) in tissue sections was never observed to undergo any changes on borohydride treatment. On the basis of these findings a very simple histochemical test has been worked out to check directly in the tissue section whether or not an observed fluorescence is due to the presence of the reacting monoamines.

Extracorporeal Albumin Dialysis in Liver Failure with MARS and SPAD: A Randomized Crossover Trial

2021 ◽  
pp. 1-8
Author(s):  
Grégoire Wallon ◽  
Cécile Guth ◽  
Céline Guichon ◽  
Sylvie Thevenon ◽  
Mathieu Gazon ◽  
...  
Keyword(s):  
Bile Acid ◽  
Liver Failure ◽  
Total Bilirubin ◽  
Surgical Intensive Care Unit ◽  
Factor V ◽  
Albumin Dialysis ◽  
Surgical Intensive Care ◽  
Dialysate Flow ◽  
Molecular Adsorbent Recirculating System ◽  
Conjugated Bilirubin

<b><i>Introduction:</i></b> Liver failure is associated with hepatic and extrahepatic organ failure leading to a high short-term mortality rate. Extracorporeal albumin dialysis (ECAD) aims to reduce albumin-bound toxins accumulated during liver failure. ECAD detoxifies blood using albumin dialysis through an artificial semipermeable membrane with recirculation (molecular adsorbent recirculating system, MARS) or without (single-pass albumin dialysis, SPAD). <b><i>Methods:</i></b> We performed a randomized crossover open trial in a surgical intensive care unit. The primary outcome of the study was total bilirubin reduction during MARS and during SPAD therapies. The secondary outcomes were conjugated bilirubin and bile acid level reduction during MARS and SPAD sessions and tolerance of dialysis system devices. Inclusion criteria were adult patients presenting liver failure with factor V activity &#x3c;50% associated with bilirubin ≥250 μmol/L and a complication (either hepatic encephalopathy, severe pruritus, or hepatorenal syndrome). For MARS and SPAD, the dialysis flow rate was equal to 1,000 mL/h. <b><i>Results:</i></b> Twenty crossovers have been performed. Baseline biochemical characteristics (bilirubin, ammonia, bile acids, creatinine, and urea) were not statistically different between MARS and SPAD. Both ECAD have led to a significant reduction in total bilirubin (−83 ± 67 μmol/L after MARS; −122 ± 118 μmol/L after SPAD session), conjugated bilirubin (−82 ± 61 μmol/L after MARS; −105 ± 96 μmol/L after SPAD session), and bile acid levels (−64 ± 75 μmol/L after MARS; −56 ± 56 μmol/L after SPAD session), all nondifferent comparing MARS to SPAD. <b><i>Conclusion:</i></b> A simple-to-perform SPAD therapy with equal to MARS dialysate flow parameters provides the same efficacy in bilirubin and bile acid removal. However, clinically relevant endpoints have to be evaluated in randomized trials to compare MARS and SPAD therapies and to define the place of SPAD in the liver failure care program.

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.

Porcine Repeat Element DNA: In Situ Detection of Xenotransplanted Cells

1995 ◽  
Vol 4 (2) ◽  
pp. 253-256 ◽  
Author(s):  
Henry F. Oettinger ◽  
Amelie Rodrigue-Way ◽  
Joyce J. Bousquet ◽  
Albert S.B. Edge
Keyword(s):  
Southern Blotting ◽  
Host Tissue ◽  
Repeat Element ◽  
Tissue Analysis ◽  
Specific Staining ◽  
Tetrazolium Chloride ◽  
Tissue Sections ◽  
Blue Tetrazolium Chloride ◽  
In Situ Detection

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated anti-digoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/ NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Applications of this technique include detection of transplanted cells or organs across a variety of xenogeneic barriers.

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