Evaluation of Decalcification Techniques for Rat Femurs Using HE and Immunohistochemical Staining

Liu, Haixia; Zhu, Ruyuan; Liu, Chenyue; Ma, Rong-Na; Wang, Lili; Chen, Beibei; Li, Lin; Niu, Jianzhao; Zhao, Dandan; Mo, Fangfang; Fu, Min; Brὂmme, Dieter; Zhang, Dongwei; S, Gao

doi:10.1155/2017/9050754

Cited by 79 publications

(94 citation statements)

References 15 publications

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“…In terms of ease of sectioning and maintenance of tissue morphology, both FA and EDTA were comparable. However, the duration for decalcification using FA was about four times less than that of EDTA, consistent with previous reports (Prasad and Donoghue, 2013;Liu et al, 2017). Our results showed that antigen retrieval using heat, trypsin, chondroitinase plus hyaluronidase and pepsin showed better staining intensity with EDTA processed sections when assessed qualitatively.…”

Section: Discussionsupporting

confidence: 92%

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

et al. 2019

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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.

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Section: Discussionsupporting

confidence: 92%

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

et al. 2019

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“…While intact hind paws provided the best context for the location of Meissner-like corpuscles across the plantar skin, they presented the greatest challenge for optimization of tissue preparation, chemical processing and staining. The size and density of tissue samples is known to positively correlate with the rate of tissue processing [25,27,30]. The volume of the mouse hind paw is at least ten times larger than its skin alone, and therefore a greater challenge for processing.…”

Section: Discussionmentioning

confidence: 99%

“…EDTA is a standard decalcifying agent that results in good antigenic preservation [25,27,33]. In comparison to decalcifiers such as formic acid (found in Cal-Ex™), EDTA is a more gentle but slower chelating agent.…”

Section: Discussionmentioning

confidence: 99%

“…To ensure enough reagent was present for complete processing, the volume of all chemicals used was at least 20 times the tissue volume. As exposure to fixation and decalcification reagents can negatively impact tissue antigenicity, our goal was to enable serial sectioning of the tissues while minimizing these processing times [25,27,28].…”

Section: Chemical Processingmentioning

confidence: 99%

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Serial Histological Sectioning of the Intact Mouse Hind Paw for Quantification of the Anatomical Distribution of Meissner-like Corpuscles across the Skin

Wai¹,

Vickaryous²,

Bent³

et al. 2020

J Histol Histopathol

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Background: Mechanoreceptors at the terminal ends of afferent nerves in the foot sole are critical for gait and balance. While previous studies have mapped afferent receptive fields and innervation densities across the foot sole using microneurography, the location and distribution of mechanoreceptors in the foot sole skin is unknown. In humans, histological investigations are limited to specimens obtained during amputation or embalmed tissues. These can be difficult to obtain and the latter poorly preserved at the extremity of the foot sole. Mice serve as an appropriate animal model to study mechanoreceptors whose function is conserved across mammalian species, however serial sectioning of intact mouse hind paws is challenging due to the dense bones and variety of soft tissues present. Consequently, digits and footpads are typically isolated for analysis and inferences made regarding mechanoreceptor density across the whole hind paw. There are no published protocols for serial histological sectioning of mouse hind paws and attempts at serially sectioning intact rat hind paws have been unsuccessful. Methods: We conducted eight experiments optimizing tissue preparation, chemical processing and sectioning techniques to achieve serial sectioning and staining of intact mouse hind paws. Two additional approaches (isolated hind paw skin and footpad biopsies) were compared to the intact hind paw approach to determine the optimal method for mechanoreceptor visualization and localization. Results: The optimized protocol for serial sectioning of intact hind paws included five days of fixation in 4% paraformaldehyde, ten days of decalcification in Cal-Ex™️ II, a 9-hour automated tissue processing protocol, embedding in a special formulated paraffin, and sectioning with specific techniques. Of the three approaches, intact hind paws provided the most context to structures visualized in the hind paw skin and thus, was the recommended protocol for future studies. Meissner-like corpuscles were located in the footpads and most abundantly in the footpads adjacent to digits II to V of the hind paw. Conclusion: The method for serial sectioning of the intact hind paws outlined in this study will allow future analysis of mechanoreceptor distribution and density in transgenic or disease mouse models.

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“…He staining. HE staining was conducted according to routine protocols 41 . Briefly, lung tissues were dissected, fixed in 4% paraformaldehyde, embedded with paraffin after dehydration, and cut into 2.5-µm thick cross-sections.…”

Section: Methodsmentioning

confidence: 99%

Comparative analysis of pathophysiological parameters between emphysematous smokers and emphysematous patients with COPD

Bai

Wang

et al. 2020

Sci Rep

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emphysematous smokers with normal spirometry form a considerable proportion of the clinical population. However, despite presenting with respiratory symptoms and activity limitation, they cannot be diagnosed with chronic obstructive lung disease (copD) according to current criteria. thus, we aimed to determine whether emphysema in smokers has a different pathogenesis from that in patients with COPD. We compared 12 pairs of lung tissue samples from emphysematous patients with normal spirometry and copD, and determined the degree of emphysema using computed tomography. With a focus on COPD-related pathogenesis, we independently assessed inflammatory response, protease-antiprotease balance, oxidative stress, and apoptosis in both groups. Both groups showed similar pathological changes at a comparable degree of emphysema; the expression of inflammatory factors was comparable, with overexpression of proteases and decreased levels of antiproteases. Moreover, there was no significant difference in the activities of glutathione and superoxide dismutase, and expression of apoptosis-related factors. in conclusion, emphysema in smokers with normal spirometry and in patients with COPD had similar pathogenesis. Forced expiratory volume in 1 second cannot be used as the sole diagnostic criterion in patients with copD; early intervention is of great importance to such patients. Chronic obstructive lung disease (COPD) is a chronic respiratory airway disease, with symptoms such as cough, sputum, and shortness of breath 1. The two most prominent pathological changes associated with COPD are the structural destruction of lung tissue and airway remodelling 2. Currently, spirometry is the sole diagnostic criterion for COPD 1,3. Forced expiratory volume in 1 second (FEV1) primarily reflects airflow obstruction and is therefore not suitable for assessing COPD symptoms such as emphysema 4-8. Studies have shown that there is a poor correlation between FEV1 and COPD symptoms 5,6 , as well between FEV1 and the degree of emphysema, as assessed by computed tomography (CT) 7,8. Patients with COPD show high heterogeneity in terms of clinical symptoms, structural destruction, and airway damage 9,10. In some patients, structural destruction of lung tissue is more prominent, and their emphysema severity develops progressively 11. These patients are termed emphysema phenotype of COPD 12,13 , and their structure destruction can be assessed with chest CT scans 14,15. However, chest CT scans have revealed that a considerable number of smokers with obvious emphysema and lung tissue damage show preserved pulmonary function 14-16. According to the current diagnostic criteria, such emphysematous smokers cannot be diagnosed with COPD. If they have same pathogenesis as patients with emphysematous COPD, appropriate interventions to reduce the clinical symptoms and block emphysema progression would be challenging in emphysematous smokers 5,17. In this study, we hypothesized that emphysematous smokers with normal spirometry and emphysematous patients with COP...

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Evaluation of Decalcification Techniques for Rat Femurs Using HE and Immunohistochemical Staining

Cited by 79 publications

References 15 publications

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

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

Serial Histological Sectioning of the Intact Mouse Hind Paw for Quantification of the Anatomical Distribution of Meissner-like Corpuscles across the Skin

Comparative analysis of pathophysiological parameters between emphysematous smokers and emphysematous patients with COPD

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