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 In vitro autoradiographic visualization of occupied estrogen receptors in the rat brain with an iodinated estrogen ligand.

1993 ◽  
Vol 41 (9) ◽  
pp. 1279-1290 ◽  
Author(s):  
M J Walters ◽  
T J Brown ◽  
R B Hochberg ◽  
N J MacLusky
Keyword(s):  
Estrogen Receptors ◽  
Computer Assisted ◽  
Physiological Changes ◽  
Tissue Sections ◽  
Receptor Complexes ◽  
Saturation Binding ◽  
Cell Groups ◽  
Cryostat Sections

Methods have been developed for the selective measurement of occupied estrogen receptors (ER) in brain tissue sections. Cryostat sections of unfixed tissue were incubated with radiolabeled estrogen at physiological temperatures, displacing endogenous receptor-bound estrogen by radioligand and thereby allowing the receptor complexes to be visualized autoradiographically after washing to remove nonspecifically bound steroid. The resultant autoradiographs were analyzed by computer-assisted densitometry. Synthetic 11 beta-methoxy-substituted radiolabeled estrogens gave the best autoradiographic images, as a result of reduced nonspecific labeling, although [3H]-estradiol was also used successfully. With the synthetic ER ligand 11 beta-methoxy 16 alpha-[125I]-iodo-estradiol, exposure times of less than 24 hr generated acceptable autoradiographs; with 3H-labeled estrogens, exposures of 3 months or more may be required. The method is sufficiently sensitive to detect physiological changes in ER occupation and to allow determination of receptor affinities and saturation binding capacities in discrete cell groups identified in sections from individual animals.

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 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 DIFFERENTIAL DEMONSTRATION OF MUSCLE AND HEART TYPE LACTIC DEHYDROGENASE OF RAT MUSCLE AND KIDNEY

1967 ◽  
Vol 15 (1) ◽  
pp. 21-31 ◽  
Author(s):  
PAUL J. MCMILLAN
Keyword(s):  
Cytochrome Oxidase ◽  
Incubation Medium ◽  
Preliminary Report ◽  
Reaction Rate ◽  
Lactic Dehydrogenase ◽  
Histochemical Demonstration ◽  
Oxidase Inhibitor ◽  
Semipermeable Membrane ◽  
Phenazine Methosulfate ◽  
Product Diffusion

A method for the histochemical demonstration of lactic dehydrogenase (LDH) is presented. This method utilizes a semipermeable membrane to minimize enzyme diffusion and high tetrazole concentration with a controlled reaction rate to minimize product diffusion. It was demonstrated that phenazine methosulfate could be used to make the reaction independent of endogenous diaphorase providing either a cytochrome oxidase inhibitor or strict anaerobiosis is employed. Differentiation of cells containing predominantly H-LDH from cells containing predominantly M-LDH was achieved by the use of 4 molal urea or pyruvate in the incubation medium. A preliminary report of the staining patterns observed in rat gastrocnemius and kidney indicates the usefulness of the technique. A new avenue to the study of the distribution of the LDH subunits is thus opened.

Respirometry for determination of the influents SS-concentration

1996 ◽  
Vol 33 (1) ◽  
pp. 311-323 ◽  
Author(s):  
A. Witteborg ◽  
A. van der Last ◽  
R. Hamming ◽  
I. Hemmers
Keyword(s):  
Experimental Design ◽  
Activated Sludge ◽  
Substrate Concentration ◽  
Full Scale ◽  
Activated Sludge Process ◽  
Respiration Rates ◽  
On Line ◽  
Large Measurement ◽  
Set Up

A method is presented for determining influent readily biodegradable substrate concentration (SS). The method is based on three different respiration rates, which can be measured with a continuous respiration meter which is operated in a cyclic way. Within the respiration meter nitrification is inhibited through the addition of ATU. Simulations were used to develop the respirometry set-up and decide upon the experimental design. The method was tested as part of a large measurement programme executed at a full-scale plant. The proposed respirometry set-up has been shown to be suitable for a semi-on-line determination of an influent SS which is fully based on the IAWQ #1 vision of the activated sludge process. The YH and the KS play a major role in the principle, and should be measured directly from the process.

Determination of trace concentrations of copper by FIA-FAAS after preconcentration on chelating sorbents

1989 ◽  
Vol 54 (7) ◽  
pp. 1785-1794 ◽  
Author(s):  
Vlastimil Kubáň ◽  
Josef Komárek ◽  
Zbyněk Zdráhal
Keyword(s):  
Sampling Frequency ◽  
Copper Determination ◽  
Flow Rates ◽  
Sample Injection ◽  
Injection Flow ◽  
Chelating Sorbents ◽  
Set Up ◽  
Trace Concentrations ◽  
Better Than

A FIA-FAAS apparatus containing a six-channel sorption equipment with five 3 x 26 mm microcolumns packed with Spheron Oxin 1 000, Ostsorb Oxin and Ostsorb DTTA was set up. Combined with sorption from 0.002M acetate buffer at pH 4.2 and desorption with 2M-HCl, copper can be determined at concentrations up to 100, 150 and 200 μg l-1, respectively. For sample and eluent flow rates of 5.0 and 4.0 ml min-1, respectively, and a sample injection time of 5 min, the limit of copper determination is LQ = 0.3 μg l-1, repeatability sr is better than 2% and recovery is R = 100 ± 2%. The enrichment factor is on the order of 102 and is a linear function of time (volume) of sample injection up to 5 min and of the sample injection flow rate up to 11 ml min-1 for Spheron Oxin 1 000 and Ostsorb DTTA. For times of sorption of 60 and 300 s, the sampling frequency is 70 and 35 samples/h, respectively. The parameters of the FIA-FAAS determination (acetylene-air flame) are comparable to or better than those achieved by ETA AAS. The method was applied to the determination of traces of copper in high-purity water.

Extra-Column Effects in Determination of Rate Parameters by the Chromatographic Method

1996 ◽  
Vol 61 (6) ◽  
pp. 844-855 ◽  
Author(s):  
Olga Šolcová ◽  
Petr Schneider
Keyword(s):  
Chromatographic Method ◽  
Dynamic Method ◽  
Axial Dispersion ◽  
Transport Parameters ◽  
Porous Particles ◽  
Loop Detector ◽  
Single Pellet ◽  
Set Up ◽  
Extra Column Effects

It was shown that the sampling loop, detector and connecting elements in the chromatographic set-up for determination of transport parameters by the dynamic method significantly influence the response peaks from columns packed with porous or nonporous particles. A method, based on the use of convolution theorem, was developed which can take these effects into account. The applicability of this method was demonstrated on the case of axial dispersion in a single-pellet-string column (SPSR) packed with nonporous particles. It is possible to handle also responses from columns packed with porous particles by a similar procedure.

scholarly journals Rapid catabolism of tyrosine-O-sulphated proteins and the formation of free tyrosine O-sulphate as an end product in rat embryo fibroblasts

1987 ◽  
Vol 243 (2) ◽  
pp. 555-559 ◽  
Author(s):  
M C Liu ◽  
M Suiko ◽  
F Lipmann
Keyword(s):  
Incubation Medium ◽  
Fresh Medium ◽  
Rat Embryo ◽  
Time Points ◽  
Rate Of Increase ◽  
Embryo Fibroblasts

Rat embryo fibroblasts, line 3Y1, were prelabelled for 24 h with [35S]sulphate and incubated in fresh medium without [35S]sulphate. A rapid efflux of the overall 35S-labelled compounds from the cells into the medium was observed. After 9 h of incubation, about 50% of the total 35S radioactivity appeared in the medium and up to 84.3% did so at the end of a 48 h incubation. Determination of [35S]sulphated macromolecules present in both the cell-associated and the incubation-medium fractions at different time points during incubation indicated that the majority of the 35S-labelled compounds released from the cells were low-Mr products derived from digestion of the [35S]sulphated macromolecules. Further analysis for tyrosine-O-[35S]sulphated proteins, which constituted only a small fraction of the overall [35S]sulphated macromolecules, showed that, after 9 h of incubation, there was a 65% decrease in the cell-associated fraction, and only 16.4% remained after 48 h. During that time, an amount equivalent to 20.7% of the cell-associated tyrosine-O-[35S]sulphated proteins originally present was released into the medium. Free tyrosine O-[35S]sulphate was generated in the cells and excreted into the incubation medium. Its rate of increase with time, however, was slow, and could account for only 12.4% of the tyrosine-O-[35S]sulphated proteins catabolized at the end of the 48 h incubation.

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