Uso de Hidróxido de Sodio como Alternativa en Técnica de Diafanización de Diente / Use of Sodium Hydroxide as an Alternative in Tooth Clearing Technique

Con el objetivo de conservar material cadavérico, se han creado diferentes técnicas y/o soluciones donde una técnica es la diafanización dental para estudiar la morfología interna del diente. Esta técnica consta en trasparentar el tejido calcificado del diente haciendo visible los conductos radiculares al inyectar una mezcla colorante en ellos. Se han descrito diferentes variantes de la técnica de diafanización como la técnica de Okumura y la técnica de Robertson, pero ambas técnicas utilizan reactivos tóxicos o de difícil acceso, por lo que se ha realizado una búsqueda de reactivos de bajo costo y fácil acceso para realizar la técnica de diafanización, reportándose que la técnica de diafanización por maceración con KOH es válida para diafanizar dientes. El objetivo del presente estudio fue utilizar NaOH en la técnica de diafanización dental por maceración, como una variante de KOH al ser una base de similar característica que el KOH. Se utilizaron 13 dientes (siete tercer molares, cinco premolares y un canino) para realizar tres variantes de la técnica de diafanización: técnica de Robertson, por maceración con KOH y por maceración con NaOH utilizando barra agitadora y agitador magnético en los dientes. Con la técnica de Robertson se obtuvo un diente completamente transparentado, mientras que los dientes diafanizados por maceración, tanto con KOH y NaOH, se transparentaron menos, aunque se hicieron visibles los conductos radiculares, por lo que el uso de NaOH en la técnica de diafanización por maceración es válida, aunque requiere más tiempo que la maceración por KOH.

SUMMARY: To preserve cadaveric material, different techniques, and solutions have been created where one technique is dental diaphanization to study the internal morphology of the tooth. This technique consists of making the calcified tooth tissue transparent and making the root canals visible by injecting a dye mixture into them. Different variants of the diaphanization technique have been described, such as the Okumura and the Robertson techniques. However, both techniques use toxic or difficult-to-access reagents, so a search has been made for low- cost and easily accessible reagents to perform the diaphanization technique, reporting that the diaphanization technique by maceration with KOH is valid for the diaphanization of teeth. This study aimed to use NaOH in the dental clearing technique by maceration as a variant of KOH since it is a base with similar characteristics to KOH. Thirteen teeth (seven third molars, five premolars, and one canine) were used to perform three variants of the diaphanization technique: Robertson technique, KOH maceration, and NaOH maceration using a stirring bar and magnetic stirrer on the teeth. With the Robertson technique, a completely transparent tooth was obtained, while the teeth cleared by maceration, with both KOH and NaOH, were less transparent, although the root canals became visible. Therefore, using NaOH in the diaphanization technique by maceration is valid, although it requires more time than KOH maceration.

A comparison of three decalcification agents for assessments of cranial fracture histomorphology.

The extraction of mineral calcium from bone by decalcification is a critical step in the preparation of histological samples for light microscopy. This study assessed the time required for complete decalcification and the resultant histomorphological preservation of bone histomorphology by three decalcification agents: 7% hydrochloric acid (HCl), 5% nitric acid, and 10% ethylenediaminetetraacetic acid (EDTA). The goal of this study was to identify which decalcification agent provides the optimal combination of expedient processing and quality histological outcomes of cranial fracture samples. HCl provided the most rapid decalcification ( X ¯  = 3.57 days), nitric acid followed closely ( X ¯  = 10.35 days), while EDTA took significantly longer on average ( X ¯  = 78.97 days) but encompassed a broader range of times. Decalcification agent, sample thickness, sample width, and decedent age are significant predictors of decalcification time. Sample visualization quality, measured for tissues, cells, and nuclei on a five-point Likert scale, was highest for samples decalcified in 10% EDTA, second highest using 5% nitric acid, and lowest for 7% HCl. The quality difference between EDTA and nitric acid was not highly significant for any of the three features. For basic assessments of bone histomorphology, the study results indicate 5% nitric acid is suitable for the decalcification of adult specimens and samples thicker than 3 mm. EDTA is a suitable agent for thin samples of the cranial vault (<3 mm) from infants and young children less than three years old, decalcifying samples in a timeframe comparable to nitric acid while providing the best quality and clarity of samples.

[From an optimal management of bone tissue samples to a quality patients' care in 2022 : A new paradigm]. / D'une gestion optimale des échantillons osseux à une prise en charge de qualité des malades en 2022 : un nouveau paradigme.

Bone tissue can be involved by primitive or metastatic tumors and requires a specific processing both at the department of pathology and during multidisciplinary meetings. The development of fine-needle percutaneous biopsies and of molecular techniques in bone tumor pathology requires a specific management. Moreover, decalcification of samples is crucial but can be deleterious if not controlled or not appropriate. The aim of this review is to provide recommendations for management and decalcification of bone tumor samples.

Evaluation of EDTA and nitric acid solutions for decalcification of joints in AG/WT, BALB/c, C57, DBA1/J mice, and in Wistar rats.

Decalcification of mineralized samples for microscopic analysis involves competing factors including decalcification time, preservation of tissue integrity and cost. We investigated the utility of different decalcification solutions for studying joints in AG/WT, BALB/c, C57, DBA1/J mice and Wistar rats. The hind paws of the rodents were removed and fixed with 10% buffered formalin. Specimens were divided randomly into three groups for demineralization: 10% nitric acid, 12.5% EDTA at room temperature and 12.5% EDTA at 35 °C with shaking. Sections of joints were stained with hematoxylin and eosin (H & E). We evaluated decalcification time and expense, ease of cutting sections, preservation of nuclear basophilia and intranuclear detail, and intensity of eosin staining. The 10% nitric acid solution produced the most rapid decalcification for the mice, but not the rats. The 12.5% EDTA solution at 35 °C with shaking did not decrease decalcification time. Effects on microtomy were variable as were the effects on H & E staining. The EDTA solution provided the best basophilia and intranuclear detail for the mice. For rats, only 12.5% EDTA at 35 °C with shaking produced good preservation. Preservation of nuclear basophilia and intranuclear detail for rats was best with 10% nitric acid and EDTA 35 °C. For mice, 10% nitric acid failed to preserve nuclear basophilia and intranuclear detail. For intensity of eosin staining, EDTA at room temperature and EDTA 35 °C was best for both mice and rats. Sections also exhibited good H & E staining in most samples decalcified with 10% nitric acid. Although we found considerable variation among groups of animals, we found less variation among the different mouse strains than between mice and Wistar rats.

Optimization of ovine bone decalcification for increased cellular detail: a parametric study.

There are many published methods of decalcifying bone for paraffin histology; however, the current literature lacks details regarding the processing of ovine tissue. Ovine bone tissue presents challenges, as samples are often denser and larger than other comparative animal models, thus increasing decalcification times. Trifluoroacetic Acid (TFAA) has previously been used to decalcify ovine bone samples for histological analysis. Unfortunately, TFAA is a strong acid and often results in loss of cellular detail, especially in the connected soft tissue. This is generally manifested as over staining with eosin, and a decrease in cellular features which impacts overall histological interpretation. It is well known that leaving tissue in acid for long periods degrades cellular detail; therefore, minimizing decalcification time is critical to accurately determine cellular morphology. Six decalcification solutions (8% TFAA, 20% TFAA, 8% formic acid, 20% formic acid, Formical-4, and XLCal, and three temperatures (room temperature, 30°C, 37°C), were examined to determine their effects on cellular detail in ovine vertebrae and humeral heads. These data clearly indicate that 20% formic acid at 30°C yielded better decalcification rates (2.6 d ± 0.9 d) and cellular detail (none to mild changes) for the vertebrae samples, and 20% formic acid at RT yielded the best cellular detail (none to minimal loss) for humerus samples with a moderate amount of time (6.5 d ± 1.7). These results should establish the optimal demineralization procedures for ovine bone used in scientific studies resulting in improved cellular detail while minimizing decalcification times.

Optimization of decalcification techniques for histologic examination of the rat maxillary and mandibular incisors for toxicity studies.

The objective of this study was to provide optimized processing for examination of rat incisors in nonclinical toxicity studies that enables analysis using immunohistochemistry (IHC). Rat maxillas and mandibles were decalcified in Immunocal, a formic acid decalcifier, and Decal Stat, a hydrochloric acid decalcifier, to evaluate tissue quality when with hematoxylin and eosin (H&E) stain and an IHC. Following necropsy of 10 to 13-week-old male Sprague Dawley rats, tissues were collected, trimmed, fixed in neutral buffered formalin (NBF), and placed into the corresponding decalcifying solution. After a pilot study with multiple timepoints for both decalcifying solutions, times were selected for the definitive study. Incisors in the definitive study were decalcified for 72, 96 or 120 hours in Immunocal and 24 hours in Decal Stat, trimmed, processed, embedded in paraffin, and sectioned. The microtomy process and sections were evaluated by histotechnologists. Sections were stained withH&E or an IHC to detect vimentin. Veterinary pathologists used blinded assessment to evaluate staining and tissue quality. The H&E sections from Immunocal timepoints scored higher based on criteria such as cellular morphology. However, tissue quality decreased at 120 hours with Immunocal but was adequate after 24 hours with Decal Stat. For IHC, moderate to excellent expression of vimentin was observed at timepoints for both decalcifiers. Optimal tissue sectioning and histological quality were achieved on incisor sections decalcified for 96 hours with Immunocal and 24 hours with Decal Stat.

Cracking it - successful mRNA extraction for digital gene expression analysis from decalcified, formalin-fixed and paraffin-embedded bone tissue.

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

Effect of EDTA decalcification on estrogen receptor and progesterone receptor immunohistochemistry and HER2/neu fluorescence in situ hybridization in breast carcinoma.

Bone is the most common site of metastasis in breast carcinoma (BC). Treatments for metastatic BC depend on various factors, including the tumor's estrogen receptor (ER), progesterone receptor (PR), and HER2 status. Bone biopsies require decalcification which may affect the accuracy of ER and PR immunohistochemistry (IHC) and HER2 situ hybridization (FISH) studies. Ethylenediaminetetraacetic acid (EDTA) decalcifying solutions have been theorized to have no significant impact on ER and PR IHC or HER2 FISH analyses. We completed a prospective study of the effect of EDTA decalcification on ER and PR IHC and HER2 FISH in 29 cases of BC. Samples from 29 BC resections were collected and formalin fixed between 12 and 24 h. Control samples were routinely processed, whereas test samples were placed in EDTA for 48 h. ER and PR slides were blinded, randomized, and evaluated. Blinded samples underwent HER2 FISH assays where an average HER2 copy number and HER2/CEP17 ratio were calculated. Paired differences between EDTA and control samples were compared for ER and PR positivity, average HER2 copy number, and HER2/CEP17 ratios using paired-samples t-tests (PST) and Wilcoxon signed-rank test (WSR). PST and WSR tests yielded no significant difference between EDTA and control tissue for ER% (PST: P = 1; WSR: P = 0.916), PR% (PST: P = 0.973; WSR: P = 0.984), HER2 copy number (PST: P = 0.124; WSR: P = 0.103), and HER2/CEP17 ratio (PST: P = 0.25; WSR: P = 0.105). The use of EDTA in bony tissue is therefore a valid decalcification method to ensure accurate assessment of ER and PR IHC and HER2 FISH in metastatic BC.

Patterns of brachyury expression in chordomas.

INTRODUCTION: Chordomas are rare malignant midline tumors, presumed to arise from notochordal remnants. This was further suggested by the discovery of the brachyury in chordomas pathogenesis. Its immunohistochemical expression has become the principal adjunct in the diagnosis of chordomas. However, studies about brachyury expression in chordomas are not fully comparable, mainly because they use different primary antibodies. Thus, the aim of this study is to investigate the expression of brachyury expression in a series of chordomas in conjunction to clinicopathological characteristics and to review the relevant literature providing all the details needed in the immunohistochemical study of brachyury. MATERIALS AND METHODS: This is a retrospective study of 62 chordomas, diagnosed over a 22-year period. No dedifferentiated or poorly differentiated cases were included. A monoclonal primary antibody (clone A-4) was used and brachyury expression was evaluated by the H-score. Clinicopathological parameters studied were age, sex, tumor localization, decalcification status and tissue age. Fetal notochords were used for comparison. RESULTS: Mean H-score of nuclear brachyury expression was 129.8. The tissue age significantly influenced brachyury expression, the older samples expressing less brachyury. Decalcification demonstrated a trend to weaken brachyury expression. Clinical characteristics were not correlated with the patterns of brachyury expression. Notochords were negative. Literature review reveals several polyclonal antibodies used and a positivity of 75%-100% in chordomas with even more variable results in notochords. CONCLUSION: In chordomas, as in other tumor types, an uniformization of studies about brachyury expression is needed, by considering the clone used, and the decalcification and the age of the sample, given the growing importance of brachyury in diagnosis and therapeutic steps.

A Simple and Quick Method for Decalcification Using Mouse Tail as a Model for Preparation of Lymphedema Study.

The disadvantage of 10% EDTA decalcification is a long time-consuming. It needs to identify a quick and straightforward decalcification method when the preparation of lymphedema models using mouse tail which was a sample of bone wrapped in other tissues. In the present study, mouse tail samples were decalcified in 10% EDTA at 25, 37, and 42°C, respectively, with continuous shaking (150 rpm/min). The histologic integrity of samples was evaluated by hematoxylin and eosin staining, and the preservation of antigenicity was tested by either immunohistochemistry or immunofluorescence. The decalcification was distinctly accelerated by temperature. Results of hematoxylin and eosin staining were similar among different temperature groups. Immunohistochemistry and immunofluorescence staining revealed almost no signals in samples decalcified at 42°C for 1 week. Clear signals were detected when samples were decalcified at 37 and 25°C.

Demineralized Murine Skeletal Histology.

Cartilage and bone are specialized skeletal tissues composed of unique extracellular matrices. Bone, in particular, has a highly calcified or mineralized matrix that makes microtomy and standard histological studies very challenging. Therefore, methods to appropriately fix and decalcify mineralized skeletal tissues have been developed to allow for paraffin processing and standard microtomy. In this chapter, we will illustrate methods for tissue grossing, fixation, decalcification, paraffin processing, embedding, sectioning, and routine histological staining of demineralized murine skeletal tissues. We will also discuss methods for decalcified frozen sectioning of skeletal tissues with and without the use of a tape-transfer system.

Immunofluorescent Staining of Adult Murine Paraffin-Embedded Skeletal Tissue.

Immunohistochemistry, or immunolabeling, is a key method for the identification of protein expression and localization. Successful detection relies on a low signal-to-noise ratio, which is affected greatly by antibody specificity as well as the staining protocol. Immunohistochemistry in the mouse is challenging, particularly in adult skeletal tissue, due to the need for long decalcification, high autofluorescence and high levels of endogenous peroxidase. Here, we describe a highly sensitive protocol for protein detection in decalcified paraffin-embedded sections from adult mouse skeletal tissue. By using four levels of amplification, this method allows for the identification of even low-abundance proteins.

Is it possible to detect lead derived from gunshot residues on decalcified human bone by means of a histochemical staining with sodium rhodizonate?

In this study we have evaluated the detectability of lead deriving from Gunshot Residues (GSR) on human bone using colorimetric techniques as well as the possible interferences of decalcification on the stainability of these particles. 22 well preserved cadavers were divided into two groups: group A enlisted 11 victims deceased because of gunshot injuries (bones and soft tissues sampled in correspondence of the entry wound of the bullet)- and group B, comprehending 11 victims deceased for natural causes (specimens sampled from intact skin areas). All the samples, after decalcification processes for bone specimens with a 14 % HCl solution, underwent standard histological procedures and were stained with hematoxylin and eosin (H&E), sodium rhodizonate (Na-R) and acidified sodium rhodizonate (Na-R-HCl 5 %). Two additional slides of soft tissues for each group were pre-treated with HCl 14 % before being stained with likewise staining. In group A, 10 samples out of 11 resulted to be negative to Na-R and Na-R-HCl 5% techniques while all the corresponding soft tissues resulted to be positive for the same staining. Additional soft tissue slides processed with 14 % HCl solution were negative in 10 cases out of 11. No sample in group B showed positivity to the staining procedures neither with nor without pre-treatment with 14 % HCl. The search for GSR residues on human bone is therefore not executable with Na-R and Na-R-HCl 5 % techniques since the essential decalcifying procedure modifies the stainability of the residues therefore adulterating the results.

Diafanización dental de cuatro especies de seláceos (Carcharhinus leucas, Galeocerdo cuvier, Rhizoprionodon longurio y Sphyrna sp) / Dental diaphonization of four salaceos species (Carcharhinus leucas, Galeocerdo Cuvier, Rhizoprionodon longurio and Sphyrna sp)

El estudio morfológico de la dentadura de chondrichthyes representa un carácter taxonómico importante empleado para la clasificación e identificación de diferentes especies. Se diafanizaron dientes de cuatro especies distintas de selacimorfos (Carcharhinus leucas, Galeocerdo cuvier, Rhizoprionodon longurio y Sphyrna sp.) con la finalidad de estandarizar una técnica dental para su transparentación. Estandarizando la técnica de Okumura-Aprile aplicada para la diafanización dental de humanos, se obtuvo una diafanización óptima en las cuatro especies en tratamiento con HCl al 7 % donde se podía observar con claridad la cámara pulpar, por lo que podemos concluir que la técnica de Okumura-Aprile es eficiente en la diafanización dental de tiburones.

The morphological study of the chondrichthyes teeth represents an important taxonomic characteristic used for the classification and identification of different species. The teeth of four different species of selacimorphs (Carcharhinus leucas, Galeocerdo cuvier, Rhizoprionodon longurio and Sphyrna sp.) were diaphonized in order to standardize a dental technique for their transparency. By standardizing the Okumura-Aprile technique applied for the dental diaphonization of humans, an optimal diaphonization was obtained in the four species treated with 7 % HCl where the pulp chamber was clearly observed. Therefore, we may conclude that the OkumuraAprile technique is efficient in shark dental diaphonization.

Whole-Tissue Immunolabeling and 3D Fluorescence Imaging to Visualize Axon Degeneration in the Intact, Unsectioned Mouse Tissues.

Axon degeneration destructs functional connectivity of neural circuits and is one of the common, key pathological features of different neurodegenerative diseases. However, conventional histochemistry methods, which largely rely on tissue sections, have intrinsic limitations in examining the 3D distribution of axonal structures on the whole-tissue level. This technical shortcoming has continuously impeded our in-depth understanding of pathological axon degeneration in many scenarios. To overcome such drawback encountered in the research field, we describe here a general protocol of whole-tissue immunolabeling and 3D fluorescence imaging technique to visualize axon degeneration in the intact, unsectioned mouse tissues. In particular, experimental steps of tissue harvesting, whole-tissue immunolabeling, tissue optical clearing, and 3D fluorescence imaging have been systematically optimized, which makes the protocol effective for assessing integrity of the axonal structures in a variety of tissues. Notably, it has enabled the 3D fluorescence imaging of chemotherapy- or traumatic injury-induced axon degeneration within the bones (e.g., femurs) or bone-containing tissues (e.g., hindpaws), which had previously been inaccessible to conventional histochemistry methods. This protocol is therefore readily compatible with many areas of the research on axon degeneration and is poised to serve the field in future investigations.

Histological, Histomorphometrical, and Biomechanical Studies of Bone-Implanted Medical Devices: Hard Resin Embedding.

The growing incidence of degenerative musculoskeletal disorders as well as lifestyle changes has led to an increase in the surgical procedures involving implanted medical devices in orthopedics. When studying implant/tissue interface in hard materials (i.e., metals or dense plastics) and/or in large bone segments, the hard plastic embedding of the intact undecalcified tissue envelope with the implant in situ is needed. The aim of this work is to describe the advances and the possibilities of high-temperature methyl methacrylate (MMA) embedding for the histological, histomorphometrical, and biomechanical assessment of bone-implanted medical devices. Unlike routine techniques, undecalcified bone processing histology, using high-temperature MMA, requires a complex and precise sample processing methodology and the availability of sophisticated equipment and software for both sample preparation and analyses. MMA embedding permits the evaluation of biological responses to the presence of implanted medical devices without implant removal, allowing simultaneous qualitative and quantitative histological evaluation, both static and dynamic histomorphometry, and biomechanical analyses not possible with tissue decalcification. MMA embedding, despite being a demanding procedure, is still preferred to other kinds of resin-based embedding because of its peculiar characteristics, which allow the study of samples of big dimensions also implanted with hard materials without reducing the sample or removing the material. Dynamic measurements are allowed together with biomechanical investigations at the bone-biomaterial interface, obtaining a comprehensive and precise evaluation of the safety and effectiveness of medical devices for orthopedic regenerative, reconstructive, and reparative surgery.

The structure of cortical bone as revealed by the application of methods for the calcified matrix study.

Calcination and decalcification are basic procedures useful to a morphological approach of a biological, composite material like cortical bone. The study was carried out on a whole human femur conserved in liquid (from an educational collection). Cortical fracturing and SEM observation of vascular canals surface collagen texture was used to study bone deproteination at scalar temperatures (400-1,200°C) and acid bone decalcification at crescent time intervals. Heating burned and vaporized the organic matrix with shrinkage of the bone specimens as documented by the weight loss and transverse surface morphometry. SEM showed a pattern of aligned spherulites at 400°C which maintained the collagen fibrils layout (like a mineral cast), followed by a spherulites fusion progression with the temperature increments. At 1200°C a crystalline-like structure of tightly-packed trapezohendron units. XRD analysis supported the SEM morphology displaying the complete Debey rings of hydroxyapatite and spotted Debey rings of withlockite. Surface Ca and P elution was documented after 12 hr of exposition to the acid solution by dissolution of spherulites and the whole canal surface decalcified in depth after 15 days by SEM-EDAX analysis. The periodic pattern of collagen fibrils was still evident up to 15 days of decalcification together with fine granular deposits of a not-collagenic proteic material, while after 30 days no period was observed in the decalcified fibrils. Collagen mineral cast at 400°C calcination. Complete crystalline transformation at 1200°C. Up to 15 days of decalcification fibrils period maintained.

Effect of decalcification protocols on immunohistochemistry and molecular analyses of bone samples.

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (<5 cycles of 6 h each) did not alter antigenicity and allowed for detection of gene mutations, amplifications or even fusion transcripts. EDTA showed superiority for in situ hybridization techniques. According to these results and our institutional experience, we propose recommendations for decalcification of bone samples, from biopsies to surgical specimens.

A novel, rapid, and low cost method for preparing tissues with metallic stents for routine histology.

BACKGROUND: Coronary artery stenting has become a common procedure and cardiovascular pathology specimens containing these metallic stents are accordingly becoming common. Histologic examination of stented vessels is imperative, but special techniques are needed due to the presence of metal within the tissue. We describe a rapid and inexpensive method for preparing stented vascular specimens for routine histology suitable for use in almost any histology laboratory. DESIGN: After formalin fixation and decalcification, stented vascular segments were freeze-embedded and sectioned using a handheld power micro cutoff wheel tool into ~1 mm slices. Sections were allowed to thaw and the strut shards removed with fine forceps. No longer containing metal, the sections were processed for routine paraffin embedding, microtomy and staining. RESULTS: Histologic sections showed only minor tissue disruption around the stent struts. In our experience with 25 stented arteries (mean interval from implantation 5.6 years), the mean subjective section quality score was 4.1 out of 5. The position of each strut could easily be determined, along with neointimal in-stent restenosis and thrombosis. Local reaction to each strut could be surmised even if minor tissue disruption occurred. The entire process was completed in 2-3 days. The incremental cost over that of routine histology is nominal. CONCLUSION: This method for examining stented vascular segments histologically could readily be applied in most pathology laboratories and serves as a highly practical solution to dilemma of examining stents histologically.

Optimization of immunohistochemical detection of collagen type II in osteochondral sections by comparing decalcification and antigen retrieval agent combinations.

Bone containing tissues such as osteochondral joint are resistant to routine tissue processing, therefore require decalcification. This technique causes removal of mineral salts, but in the process may macerate the organic tissue, hence the need for tissue fixation. Such severe processing demands careful antigen retrieval to necessitate optimal staining. The aim of our study was to compare five different antigen retrieval protocols (heat retrieval and protein digestion) following decalcification of rabbit knee joints using two different techniques (20% formic acid and 10% ethylenediamine-tetra acetic acid: EDTA). Osteochondral sections were compared based on time required for decalcification, ease of sectioning, morphological integrity using HE staining and antigen preservation (Collagen type II) using immunohistochemistry. The two decalcification solutions did not impair the tissue morphology and ease of sectioning. Joints processed with formic acid decalcified four times faster than EDTA. Among the five antigen retrieval approaches, maximal collagen II uptake with minimal nonspecific staining was found with protein digestion (pronase and hyaluronidase) in both formic acid and EDTA sections. For osteo-chondral sections, we recommend using 10% EDTA for decalcification and pronase plus hyaluronidase for antigen retrieval if maintaining tissue morphology is crucial, whereas if time is of the essence, 20% FA with pronase plus hyaluronidase is the faster option while still preserving structural integrity. Clin. Anat. 33:343-349, 2020. © 2019 Wiley Periodicals, Inc.