Feulgen Reaction for Thymonucleic Acid

The importance of thymonucleic acid in tissues is discussed briefly. The technic of the Feulgen reaction which has been employed in photometric histochemical observations in tumors is described. The evidence for the specificity of the Feulgen reaction is reviewed and additional experimental observations are reported. The staining of tissues by the Feulgen reaction is compared with that of hematoxylin, basic fuchsia, and fuchsin-sulfurous-acid reagent in which the color had been developed by the addition of formaldehyde. The stains were compared with respect to (1) the selective staining of the cytologic components of the tissues, (2) the staining of tissues following varying intervals of acid hydrolysis and (3) the photometric determination of the fading of the stained tissue by a carbon arc light. The photometric apparatus employed is suitable for the study of many problems on the staining of tissues. Staining by the Feulgen reaction is different from that of both the basic fuchsin from which the fuchsin-sulfurous-acid was prepared and from that of the product of the fuchsin-sulfurous-acid which had reacted with an aldehyde. Under carefully controlled conditions, the Feulgen technic is a relatively specific histochemical reaction for thymonucleic acid.     

The Staining Of Acid Fast Bacilli In Sections Of Glycol Methacrylate Embedded Tissues

Acid-fast bacilli can be stained in tissue embedded in glycol methacrylate. Modification of the Ziehl-Neelsen technique, along with changes in the formula of the plastic embedding medium, allow production of 1 to 2 micron sections which retain their integrity throughout the procedure, and within which the bacilli are clearly visible.     

Combined In Situ Zymography, Immunofluorescence, And Staining Of Iron Oxide Particles In Paraffin-embedded, Zinc-fixed Tissue Sections

AbstractSuperparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

The Specificity of the Feulgen Reaction for Thymonucleic Acid

The results of experiments on the specificity of the Feulgen reaction for thymonucleic acid do not substantiate the observations of Carr. The staining is not localized in the nucleus because of the destruction of cytoplasmic constituents following acid hydrolysis or because of the absorbing power of chromatin, since the cytoplasm and nucleolus can still be stained by numerous dyes. The effects of factors such as the acid hydrolysis and sulfurous acid washing baths upon the cytologic distribution of dye were studied on tissues stained with (1) fuchsin-sulfurous-acid (Feulgen) reagent, (2) fuchsin-sulfurous-acid reagent colorized by the addition of formaldehyde, (3) basic fuchsin in one-tenth normal HCl, and (4) basic fuchsin in distilled water. Under comparable conditions, important differences between these stains were found in the effects of preliminary hydrolysis; rapidity of staining and destaining; extractability of dye from tissues by water, alcohol, and sulfurous acid solution; rate of fading from exposure to light; localization of stain in tissues; and differences in hue. After treating tissues with desoxyribonuclease, an enzyme which acts only upon thymonucleic acid, cells do not stain with the Feulgen technic. Following removal of nucleic acid from chromatin by hydrolysis, attempts to demonstrate an absorption of thymonucleic acid upon the residual nuclear protein were unsuccessful.

The evidence for and against the specificity is discussed. In agreement with most other investigators, on the basis of the evidence in the literature as well as these experiments, it is concluded that when properly controlled the Feulgen reaction is relatively specific for thymonucleic acid.     

Comparison Of The Efficacy,Adverse Effects,And Cost Of Zoledronic Acid And Denosumab In The Treatment Of Osteoporosis

ObjectiveInjectable osteoporosis drugs are increasing in popularity due to their efficacy and convenient administration. In this retrospective comparison of the two available treatments, denosumab (Prolia®) and zoledronic acid (ZA, Reclast®), we aimed to determine and compare the efficacy and tolerability of denosumab and ZA.MethodsThe charts of patients who received denosumab and ZA at Loyola Hospital were reviewed, and adverse events were noted. Of primary interest were myalgias, flu-like symptoms, back pain, and fractures. A questionnaire regarding the efficacy, tolerability, and treatment cost supplemented this chart review in a subset of study participants. Bone mineral density (BMD) changes, bone turnover markers, and questionnaire results were also compared.ResultsThe study cohort consisted of 107 patients (51 denosumab, 56 ZA). The denosumab group had a greater mean increase in spine BMD at 1 year (0.060 g/cm²) than the ZA group (0.021 g/cm²; P = .04). The change in femur and spine BMD at 1 year were not significantly different between the 2 groups. The ZA group had a significantly greater incidence of mild flu-like symptoms (29% ZA group vs. 0% denosumab group; P = .04).ConclusionThe denosumab group had a higher mean increase in spine BMD, and the ZA group had a higher incidence of flu-like symptoms, but the study groups were statistically similar in terms of patient satisfaction. As denosumab is still a relatively new therapy, there were a limited number of patients with posttreatment data available for comparison. As more posttherapy data become available, it can be further investigated. (Endocr Pract. 2015;21:275-279)     

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10¹⁰ viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.     

Esterification In Organic Solvents: Selection Of Hydrolases And Effects Of Reaction Conditions

Fifty different hydrolases were screened for retention of high esterification activity in an organic solvent with citronellol as substrate. Although 22 hydrolases were very active as catalysts in the organic solvent, lipase from Candida cylindracea (lipase OF 360) was selected for further examination of the effects of reaction conditions on enzyme activity, with regard to catalyst availability and activity retention after immobilization. When the enzyme was entrapped in hydrophobic polyurethane gels, water-saturated isooctane was found to be the most suitable solvent, whereas polar solvents caused reversible catalyst inactivation. Entrapment significantly enhanced the operational stability of the lipase in the organic solvent.     

The Feulgen Reaction after Glutaraldehyde Fixation

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH₄ in 1% NaH₂PO₄ for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCI for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

The Proliferative Status And Clonogenic Capacity Of Tumour Cells In A Transplantable Rhabdomyosarcoma Of The Rat Before And After Irradiation With 800 Rad Of X-Rays

R-1 Rhabdomyosarcomas Transplantable In The Inbred Strain Of Wag/Rij Rats Were Labelled Semicontinuously With ³H-Tdr And Subsequently Irradiated With X-Rays In Order To Estimate The Rate Of Recruitment Of Q Into P Cells And To Obtain More Insight Into The Importance Of Hypoxia With Respect To The P And Q Status Of Clonogenic Cells. Labelling Was Performed By Giving Animals From One To A Total Number Of Up To Seven Intraperitoneal Injections With ³H-Tdr Within 30 Hr. Half Of The Number Of Tumours Received An Acute Dose Of 800 Rad Of 300 Kv X-Rays At 45 Min After Completion Of The Last Labelling In Order To Reduce The Fraction Of Well Oxygenated Cells Among The Surviving Clonogenic Cells To Less Than 1%; Labelled Control Tumours Were Sham Irradiated. Fractions Of Labelled Tumour Cells, Li_Tumour’Were Evaluated From Autoradiographs Prepared From Sections Of Tumours That Were Fixed 15 Min After Irradiation: Fractions Of Labelled Cell Foci, Li_Fo, Were Determined In Autoradiographs Prepared From Cultures Of Cells Isolated From The Remaining Parts Of The Tumours. Analysis Of The Variation In Li_Tumour As A Function Of Time And Of The Number Of Injections Showed That The Rate Of Recruitment Of Q Cells Into P Cells Is Most Likely Less Than 1%/Hr. Comparison Of Data On Li_Fo Derived For Irradiated And Non-Irradiated Tumours Indicates That Clonogenic Q Cells Are More Frequently Present Among Cells Which Survived The Dose Of 800 Rad Of X-Rays. These Results Suggest That, In R-1 Tumours Of About 800 Mm³ The Clonogenic Q Cell Population Contains A Larger Fraction Of Hypoxic Cells Than The Fraction Of Hypoxic Cells Present In The Clonogenic P Cell Population. Several factors are discussed which complicate the analysis and may influence the results, e.g. reutilization of labelled debris of dead cells from irradiated tumours.     

Effectiveness Of Sch1ff Variants In The Periodic-Schiff and Feulgen Nucleal Technics

The presence of additional S02 in Schiff reagents above that required for bleaching of the basic fuchsin has no significant effect on the behavior of such reagents in performing the periodic-Schiff or Feulgen nucleal reactions. Schiff reagents made from aqueous solutions containing less than 0.125% basic fuchsin will not demonstrate in these reactions all the tissue elements stained by stronger solutions. Schiff reagents made from aqueous solutions containing more than 0.5% basic fuchsin will in time precipitate considerable amounts of leucofuchsin. Fresh activated charcoal discriminates much more sharply between the yellow contaminant which occurs in basic fuchsin when the fuchsin has been converted to the leuco state than it does when the fuchsin is in the colored condition. Precipitation of leucofuchsin occurs at pH 3 or slightly above in Schiff reagents of generally useful concentration, but at lower levels down to the pH of 10 N HC1, hydrogen ion concentration does not influence their histochemical effectiveness.     

Block Staining of Nervous Tissue with Silver IV. Embryos

Procedures having enhanced reliability over older methods for both Bielschowsky and Cajal types of stain are described.

Fixation of embryos in a solution containing 4% formaldehyde and 0.5% trichloracetic acid greatly improved the staining of neural elements by Bielschowsky's method.

Among the variations of Cajal's type of staining tried, a modification of Ranson's pyridin-silver method gave the most complete staining of neurofibrillar elements. Washing for 0.5 to 1 hour after silver impregnation and shortening of the reduction time from 24 to 4 hours corrected the tendency of the method to overstain.     

Fast Green Staining Of Whole Embryos For Examination And Photography

Immersion for 15 seconds in a 1:1 solution of 0.25% fast green and 95% ethyl alcohol facilitates the examination of normal and abnormal embryos for gross surface morphology and renders their surface detail highly photogenic. The method does not interfere with subsequent histological staining when the embryos are sectioned.     

Block Staining of Nervous Tissue with Silver: II. Trichloracetic Acid, Sulfosalicylic Acid, Hofker's and Carnoy's Fluids as Fixatives

Alcoholic solutions of sulfosalicylic acid, trichloracetic acid and Hofker's solution fixed mammalian spinal cord with less shrinkage than ammoniated alcohol, but during the necessary alkalinization, washing, and silvering, the acid fixed specimens shrank more than those fixed in the alkaline alcohol. Specimens fixed in Carnoy's fluid shrank most. Successful silver stains were obtained after all the fixatives.