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.     

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.     

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.     

Histochemical studies on the localization and activity of acid phosphatase in calcifying tissues

Summary Acid phosphatase activity has been studied in cold microtome sections and using simultaneous azo coupling method in developing teeth and bone, and serial sections were made for the demonstrations of alkaline phosphatase.1. In developing teeth, strongest activity of acid phosphatase was found in the distal portion of high columnar ameloblasts associated with heavy calcification in the rodent incisor, and ameloblasts and odontoblasts in adjacent occlusal surface in molar teeth. However, the activity of immatured ameloblast and crevicular aspects of molar were weaker.2. In the epiphyseal bone trabeculae a striking acid phosphatase reaction was found.3. As regards to the effects of decalcifying solutions to the enzymatic activity, the use of EDTA decalcifying agent (10% and pH 7 to 4) showed the best results. That is, a decrease of decalcifying time and a greater preservation of acid phosphatase activity.With 11 Figures in the Text     

Fixation,decalcification, and tissue processing effects on articular cartilage proteoglycans

Summary Neutral buffered 4% formaldehyde fixation for 48h preserved well the proteoglycan content of bovine articular cartilage. Neither subsequent demineralization in 10% EDTA, nor light microscopic tissue processing, reduced the hexosamine or uronic acid content of the tissue. Fixation in alcoholic solutions increased Safranin O binding as well as periodic acid Schiff reaction of the cartilage matrix as measured by microspectrophotometry. It is suggested that the enhanced staining of cartilage was due to better preservation of the glycoprotein oligosaccharides. Quaternary ammonium salts in the fixative suppressed the staining of cartilage matrix with Safranin O.     

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.     

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse’s solution, Plank-Rychlo’s solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse’s solution, 10% HCl, Plank-Rychlo’s solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse’s solution. We recommend Morse’s solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse’s solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions. Accepted: 3 January 2000     

Effects of decalcification on the membraneous labyrinth

Decalcification of the guinea-pig temporal bone was performed using a solution of 5.5% ethylenediamine tetraacetic acid (EDTA) (14 days) and 5% trichloracetic acid (4 days), respectively.Following the use of EDTA, the general cell structures were well preserved except for a slight distortion of outer hair cells in the organ of Corti. The fine morphology was acceptable though the cell membranes frequently appeared less distinct as compared with controls. Microfilaments/microtubules were preserved in the cells. The substructures of stereocilia and kinocilia can be identified.Decalcification with trichlor acetic acid caused morphological changes at the light microscopical level. The ultrastructure was extremely poor in all cells.For quantification of cochlear hair cells according to the block surface technique, both EDTA and trichlor acetic acid can be used for decalcification with good results.     

Fluorophotometric quantitation of DNA in articular cartilage utilizing Hoechst 33258

A sensitive fluorophotometric assay was developed for the measurement of DNA in articular cartilage. The tissue was digested with Proteinase K and dodecyl sodium sulfate, followed by analysis with Hoechst 33258 dye. DNA content was determined on both fresh and lyophilized material containing as little as 50 ng DNA. The results are comparable to values for other fluorophotometric and spectrophotometric methods reported in the literature. In addition, this method can be incorporated into existing methodology, allowing quantitation of specific glycosaminoglycans in the same cartilage sample in terms of DNA.     

Decalcification for histochemical demonstration of hydrolytic and oxidative enzymes

Summary A method for histochemical demonstration of hydrolytic and oxidative enzymes following decalcification was described in mature bone and tooth by neutral EDTA decalcifying solution.The decalcifying solution, 0.5 M EDTA tetrasodium salt was adjusted to neutral pH with 5 M citric acid, obtained the most sufficient results for demonstration of enzymes in decalcified tissue. The hard tissue decalcified in 30 to 40 days at 4° C exhibited a good preservation of hydrolytic and oxidative enzymes histochemically, but a few structural destruction in a long term decalcification was found in soft tissues and certain organs.     

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:     

一种快速、经济提取外周凝血基因组DNA方法的建立

目的：建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法：摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用Ⅺ法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量。并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的Ⅺ法进行比较分析。结果：最佳的匀浆条件为：39000map,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论：与常规的外周凝血提取方法相比（蛋白酶K消化法）,本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。     

New method for determining the extent of proline hydroxylation by measuring changes in the ratio of [4-3H]:[14C]proline in collagenase digests

A sensitive colorimetric assay for proteases and certain polysaccharidases is based on the digestion of proteoglycan from bovine nasal cartilage. This high-molecular-weight substrate is trapped in the interstices of a polyacrylamide gel. The gel is then dispersed as small particles. Enzymes can diffuse into these particles and digestion products can diffuse out. Following digestion, the particles are centrifuged off and the digestion products in the supernatant are quantitated by reaction with the metachromatic dye 1,9-dimethylmethylene blue or by assay of their uronic acid content with carbazole reagent. Proteases from each of the four major classes can be quantitated at levels of 1–200 ng. The method is particularly suitable for the study of cartilage proteases that degrade matrix proteoglycans.     

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.     

In vivo DNA damage in gastric epithelial cells

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.     

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.     

Patterns of distribution of phosphomono-esterases on surfaces of demineralized bone

Summary Decalcification over short periods (5 days) with MnNa₂ EDTA, MgNa₂ EDTA and EGTA according to a method described in the present paper, creates sections of high quality with simultaneous good preservation of phosphomonoesterases on bone surfaces. In fact, the enzyme distribution seems to be comparable to that obtained by using undecalcified sections.Na₂ EDTA creates, on the other hand, poor preservation of alkaline phosphatase probably due to the fact that this chelate contrary to the other chelates removes the essential metal from the protein, leaving an unstable enzyme molecule which undergoes denaturation.Decalcification over longer periods (15 days) does not influence the pattern of distribution of acid phosphatase, whereas the alkaline phosphatase reaction becomes depressed in certain surface areas. The significance of this differential distribution is discussed. It might be an indication of differential processes of bone transformations in such a way that bone surfaces corresponding to areas of enzyme reactions are depository whereas bone surfaces corresponding to areas of lack of enzyme reaction are resorptive. New experimental designs are, however, necessary before the phenomenon is fully perceived. Two different coupling agents were used in connexion with the demonstration of acid phosphatase reaction. When HPR was used as the coupler the final enzyme distribution coincided with that usually described in the literature, i.e., strong reaction of cells adjacent to resorptive surfaces and weak reaction of cells adjacent to depository surfaces. When, however, Fast dark blue R was used all surface cells reacted markedly. This method also revealed certain cell types with nuclear reaction.     

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.     

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.     

Patterns of distribution of phosphomono-esterases on surfaces of demineralized bone.

Decalcification over short periods (5 days) with MnNa2 EDTA, MgNa2 EDTA and EGTA according to a method described in the present paper, creates sections of high quality with simultaneous good preservation of phosphomonoesterases on bone surfaces. In fact, the enzyme distribution seems to be comparable to that obtained by using undecalcified sections. Na2 EDTA creates, on the other hand, poor preservation of alkaline phosphatase probably due to the fact that this chelate contrary to the other chelates removes the essential metal from the protein, leaving an unstable enzyme molecule which undergoes denaturation. Decalcification over longer periods (15 days) does not influence the pattern of distribution of acid phosphatase, whereas the alkaline phosphatase reaction becomes depressed in certain surface areas. The significance of this differential distribution is discussed. It might be an indication of differential processes of bone transformations in such a way that bone surfaces corresponding to areas of enzyme reactions are depository whereas bone surfaces corresponding to areas of lack of enzyme reaction are resorptive. New experimental designs are, however, necessary before the phenomenon is fully perceived. Two different coupling agents were used in connexion with the demonstration of acid phosphatase reaction. When HPR was used as the coupler the final enzyme distribution coincided with that usually described in the literature, i.e., strong reaction of cells adjacent to resorptive surfaces and weak reaction of cells adjacent to depository surfaces. When, however, Fast dark blue R was used all surface cells reacted markedly. This method also revealed certain cell types with nuclear reaction.