An Ion-exchange Bone Demineralization Method for Improved Time,Expense, and Tissue Preservation

Here, we describe an ethylenediaminetetraacetic acid (EDTA)-based bone demineralization procedure that uses cation-exchange resin and dialysis tubing. This method does not require solution changes or special equipment, is faster than EDTA alone, is cost-effective, and is environmentally friendly. Like other EDTA-based methods, this procedure yields superior tissue preservation than formic acid demineralization. Greater protein antigenicity using EDTA as opposed to formic acid has been described, but we also find significant improvements in carbohydrate-based histological staining. Histological staining using this method reveals cartilage layers that are not distinguishable with formic acid demineralization. Carbohydrate preservation is relevant to many applications of bone demineralization, including the assessment of osteoarthritis from bone biopsies and the use of demineralized bone powder for tissue culture and surgical implants. The improvements in time, expense, and tissue quality indicate this method is a practical and often superior alternative to formic acid demineralization:     

The Use of a Cation Exchange Resin in Decalcification

Decalcification of 2-3 mm. sections of human ribs by 5-40% concentrations of both formic and nitric acid was subjected to a controlled study. The effect of adding 1 g. of phloroglucinol and of 2.5, 5 and 10 g. of the exchange resin (Win-3000) per 100 ml. of decalcifying solution was observed after staining celloidin sections with hematoxylin and Triosin or with Giemsa stain. Electrolytically decalcified material was included for comparison also. The following conclusions were drawn: (1) The addition of resin did not appreciably shorten the decalcification time nor enhance tissue preservation and staining qualities. (2) Formic acid, 20% or less, gave results superior to nitric acid in any concentration and also superior to those obtained after the electrolytic method. (3) The addition of 1% phloroglucinol to formic acid solution improved both preservation and staining. (4) Helly's fluid fixation with all combinations gave uniformly better results than formalin fixation.     

Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling

To analyse DNA strand breaks by terminal deoxy(d)-UTP nick-end labelling (TUNEL) in calcified tissues including bones and teeth, it is important to decalcify the tissues first. However, the effects of decalcifying reagents on the integrity of DNA are largely unknown. In the present study, we evaluated the usefulness of various decalcifying reagents including 10% EDTA (pH 7.4), 5% trichloroacetic acid (TCA), 5% formic acid, 5% HCl, 10% nitric acid, Plank–Rychlo's solution, Morse's solution and K-CX solution in TUNEL staining. Mouse maxilla was selected as the experimental system. Apoptotic cells naturally occurring in the epithelium were analysed. Tissues were assessed by soft X-ray imaging to confirm complete decalcification. The time required for decalcification of the tissue was 7 days with 10% EDTA and 2 days with other decalcifiers. Decalcified tissues were stained with Methyl/Green–Pyronine Y or 4, 6-diamidino-2-phenylindole for assessment of DNA integrity. Nuclei of epithelial cells were strongly positive for both dyes after decalcification with 10% EDTA, 5% TCA, Morse's solution and 5% formic acid. The other reagents failed to retain DNA. Our results demonstrated good TUNEL staining of the maxilla treated with 10% EDTA or 5% TCA . Based on the required time for processing and the signal-noise ratio, we recommend 5% TCA as the decalcifying reagent to analyse for DNA strand breaks.     

Influence of decalcifying agents on immunoreactivity of formalin-fixed, paraffin-embedded tissue

Summary The influence of eight decalcifying agents on the immunoreactivity of formalin-fixed, paraffin-embedded tissue for immunoglobulins, lysozyme, factor VIII-related antigen and keratin was studied using the unlabelled antibody peroxidase-antiperoxidase (PAP) method. Limited studies were also performed on tissues fixed in acid-formalin mixtures. All tissues were stained using an indirect immunoperoxidase method with mouse monoclonal antibodies to IgM, kappa and lambda light chains. The results suggest that, with controlled trypsinization of sections, it was possible to obtain optimal immunostaining for all tested antigens, with adequate preservation of histological structure, after decalcification in neutral EDTA or 10% aqueous acetic or formic acid. Tissue treated with agents containing mineral acids exhibited variable immunoreactivity and impaired counterstaining.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Immunohistological optimization of detection of bromodeoxyuridine-labeled cells in decalcified tissue

Mice were injected with a range of bromodeoxyuridine (BrdU) concentrations from 0.01 mg to 10 mg, and their jaws were fixed in buffered formalin or modified Carnoy. After EDTA or formic acid decalcification, a range of DNA denaturation schedules was assessed and immunohistological detection of BrdU-containing nuclei was performed using the Sera Lab anti-BrdU antibody MAS 250b. For Carnoy-fixed tissue, denaturation in 1 N HCl for 8 min at 60 degrees C was capable of adequately detecting an injected dose of 0.05 mg but not a dose of 0.01 mg BrdU, whereas pepsin/HCl treatment gave only weak staining after injection of 1 mg BrdU. In comparison, formalin fixation required pre-treatment with 0.2-0.4% pepsin/HCl at 37 degrees C for comparable staining intensity, but could still not adequately detect a dose of 0.1 mg BrdU. There was little detectable difference in staining between EDTA- and formic acid-decalcified tissues after injection of 10 mg BrdU.     

Rapid Decalcification Using Microwaves for in Situ Hybridization in Skeletal Tissues

In situ hybridization histochemistry is the sole tool available for detecting the localization and expression of specific RNA on histological sections under various in vivo conditions. For this paper, we examined the effect of microwave exposure on the time needed for decalcification of skeletal tissues and on the preservation of sensitivity for hybridization signals. Our data show that the use of microwave decalcification reduces the decalcification period while preserving intense hybridization signals for mouse al chain of procollagen type I mRNA in osteogenic cells in bone. The use of microwave treatment to decalcify skeletal tissues may prevent delay in obtaining experimental results or the loss of signals during in situ hybridization.     

The Effect of Decalcification and Choice of Fixative on Histiocytic Iron in Bone Marrow Core Biopsies

Iron stains are often used for bone marrow core biopsies obtained by needle biopsy of the iliac crest. Because bone most be decalcified by brief treatment with acid, it is possible that an undetermined amount of stainable histiocytic iron may be lost. A study was carried out to determine whether decalcification results in loss of histiocytic iron and the effects of fixatives and the recovery of histiocytic iron in decalcified bone marrow tissue. Aspirates of bone marrow were stained for iron with Prussian blue. Because aspirate material does not require decalcification, it served as a control for the study. One hundred bone marrow biopsies and accompanying aspirates from 100 adult subjects were evaluated. Fifty bone marrow biopsies were fixed using a fixative containing mercuric chloride (B-5) and the remaining 50 were fixed in zinc-formalin. Histiocytic iron was graded as minimal, moderate or marked depending on whether less than 5, 6-10, or more than 10 iron positive histiocytes, respectively, were observed. When histiocytic iron was markedly present in aspirate material, at least moderate amounts of stainable iron were found in 22 of 25 B-5 fixed and 21 of 25 zinc-formalin fixed decalcified bone marrow. When aspirate histiocytic iron was minimal or moderate, 14 of 25 B-5 fixed and 7 of 25 zinc-formalin fixed decalcified bone marrow specimens revealed histiocytic iron. Decalcification results in decreased recovery of stainable iron, and where histiocytic iron is minimally or moderately present, B-5 fixation results in greater postdecalcification recovery. There was no significant difference in recovery when larger quantities of histiocytic iron were present prior to the decalcification step.     

Golgi-Cox Impregnation of Peripheral Zone after Coating Tissue with Egg Yolk

Conventional methods of decalcification using acids depolymerize nucleic acids. This is a serious handicap for microspectrophotometric studies on the DNA content of calcified tissues. in the investigation reported here we have evaluated different decalcifying agents for this purpose. Liver, spleen, and maxillae of adult Wistar rats were exposed to the action of nitric acid, picric acid, and EDTA.     

Effect of EDTA on cytokeratin detection in the inner ear.

Immunohistochemical studies on the epithelium of the adult inner ear are difficult to perform without decalcification of the bony capsule. In this study, we examined the effect of decalcifying agents on the immunoreactivity of various cytokeratin antigens in the cochlear duct epithelium of 2-day-old rats, allowing the comparison of fresh and decalcified specimens. Decalcification of unfixed tissue in a solution containing EDTA or EGTA and polyvinylpyrrolidone, at pH 7.4 and 4 degrees C for a maximum period of 2 days, not only preserved the antigen epitopes but even enhanced the staining intensities in comparison with fresh specimens. This enhancement effect, caused by chelating agents and found to be blocked by prior fixation with acetone, is suggested to be caused by unmasking of the antigenic epitopes.     

The Effect of Decalcification on the Microspectrophotometric Determination of Dna

Conventional methods of decalcification using acids depolymerize nucleic acids. This is a serious handicap for microspectrophotometric studies on the DNA content of calcified tissues. in the investigation reported here we have evaluated different decalcifying agents for this purpose. Liver, spleen, and maxillae of adult Wistar rats were exposed to the action of nitric acid, picric acid, and EDTA.     

An Improved Method of Decalcification

Following several experimental investigations, an improved method of decalcification has been devised. The principle of this decalcification method is to obtain complete decalcification by a mixture of as high pH as possible without diminishing the stainability of the Nissl-granules (with Einarson's progressive staining method by means of gallocyanin). This is accomplished by the help of a buffer solution of equal parts of 8 N formic acid and 1 N sodium formate (pH 2.2). After-treatment consists only in rinsing in flowing water for 24 hours. Dehydration is in alcohol (70%, 96%, 100%); cedar oil; ligroin. Embedding in paraffin follows.     

An Improved Method of Decalcification

Following several experimental investigations, an improved method of decalcification has been devised. The principle of this decalcification method is to obtain complete decalcification by a mixture of as high pH as possible without diminishing the stainability of the Nissl-granules (with Einarson's progressive staining method by means of gallocyanin). This is accomplished by the help of a buffer solution of equal parts of 8 N formic acid and 1 N sodium formate (pH 2.2). After-treatment consists only in rinsing in flowing water for 24 hours. Dehydration is in alcohol (70%, 96%, 100%); cedar oil; ligroin. Embedding in paraffin follows.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Evaluation of Analytical Techniques to Predict Viability after Cryopreservation

Preservation of plant germplasm is important to safeguard biodiversity and to store elite plants. Cryopreservation is one of the possible preservation techniques. Research for a cryopreservation protocol is often inefficient because of slow or poor regrowth of plant material. Therefore, at least one technique, that allows a quick and accurate prognosis of viability after cryopreservation, is required. We evaluated five techniques: electrolyte leakage, triphe-nyltetrazoliumchloride (TTC) staining (visual and spectrophotometrical analysis), malondialdehyde concentrations in plant tissue and a mathematical model that relates ‘water content’ to the weight of encapsulated plant material. Electrolyte leakage and TTC-staining (if visually analysed) are efficient to predict viability. Our mathematical model allows us to save time and plant material in order to develop an efficient encapsulation—dehydration protocol. All other techniques were rejected because of the high variability of the results. This is due to the variability of biochemical activity in plant tissue and the small amount of tissue used in the experiments.     

The effects of different decalcification protocols on TUNEL and general cartilage staining.

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Decalcification end point determination by X-rays and flame photometry to obtain superior quality pulpal sections

Radiography and flame photometry have been compared as means of determining the end point of decalcification in relation to minimizing pulp-dentin separation in histological sections of teeth. Eighteen homologous pairs of vervet monkey incisor teeth were decalcified in a formic-citric acid mixture. At 24 hr intervals decalcification was monitored in half of the teeth by radiography and in the other half by flame photometry. When decalcification was complete as determined by the respective methods, histological specimens were prepared and separation at the pulp-dentin interface evaluated in hematoxylin and eosin stained step serial sections. The median separation was determined for the combined group and the median test applied. There was significantly less separation in the flame photometry group and within each group significantly less separation on the side where the knife cut from dentin to pulp.     

Safranin O reduces loss of glycosaminoglycans from bovine articular cartilage during histological specimen preparation

Summary The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm³ cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sectins using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4°C. Rougly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12c days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.     

Histochemical detection of osteocalcin in normal and pathological human bone

We investigated the immunohistochemical localization of osteocalcin in demineralized, paraffin-embedded normal and pathological human bone. Acid decalcification protocols appeared to be more suitable for osteocalcin detection than mild chelating agents. In normal lamellar bone, osteocalcin was detected in osteocytes and along the lamellar bone matrix in fine granular deposits. Under pathological conditions (osteomyelitis, neoplasia), appositional bone showed immunoreactivity in osteoblasts and osteocytes but not in the provisory woven bone matrix. Intense immunoreactivity could be seen at the cell borders of osteoclasts and the bone margins of Howship lacunae. In primary bone-forming tumors, osteocalcin immunoreactivity was detected in osteoblasts and their malignant counterparts. On the basis of these results, we conclude that optimal preservation of osteocalcin is obtained through mild acid decalcifiers. Osteocalcin is deposited in bone matrix, especially that of metabolically inactive bone. In neoplasms, osteocalcin could be a marker of osteoblastic differentiation.     

Ethylenediaminetetraacetic Acid in Ammonium Hydroxide for Reducing Decalcification Time

Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new de-calcification fluid using concentrated ammonium hydroxide (NH₄OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH₄OH required an average of six days. Light microscopy indicated good retention of cellular detail.