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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., (© 2019 Wiley Periodicals, Inc.)

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.

Aim . In routine histopathology, decalcification is an essential step for mineralized tissues. The purpose of this study is to evaluate the effects of different decalcification solutions on the morphological and antigenicity preservation in Sprague Dawley (SD) rat femurs. Materials and Methods . Four different decalcification solutions were employed to remove the mineral substances from rat femurs, including 10% neutral buffered EDTA, 3% nitric acid, 5% nitric acid, and 8% hydrochloric acid/formic acid. Shaking and low temperature were used to process the samples. The stainings of hematoxylin-eosin (HE) and immunohistochemical (IHC) were employed to evaluate the bone morphology and antigenicity. Key Findings . Different decalcification solutions may affect the quality of morphology and the staining of paraffin-embedded sections in pathological examinations. Among four decalcifying solutions, 3% nitric acid is the best decalcifying agent for HE staining. 10% neutral buffered EDTA and 5% nitric acid are the preferred decalcifying agents for IHC staining. Significance . The current study investigated the effects of different decalcifying agents on the preservation of the bone structure and antigenicity, which will help to develop suitable protocols for the analyses of the bony tissue., Competing Interests: The authors declare no conflict of interests regarding the publication of this paper.

The aim of this study was to determine the possibility of immunohistochemical demonstration of various tissue antigens in paraffin sections of zinc-ethanol-formaldehyde-fixed spinal cord, surrounded by the vertebral tissues and adjacent muscles, after decalcification in formic acid, in adult (n = 5) and newborn (n = 2) Wistar rats. In the present work, the antibodies used were against: glial fibrillary acidic protein, neurofilaments, neuronal specific nuclear protein, enzyme tyrosine hydroxylase, protein of synaptic vesicles--synaptophysin and the intermediate filament protein--vimentin. Combination of fixation in zinc-ethanol-formaldehyde and decalcification in 21% formic acid solution was shown to provide an optimal preservation of the antigens studied.

Objective: To investigate the effects of 1% hydrochloric acid and 10% nitric acid on immunohistochemical staining for several antigens., Methods: Fifteen cases of focally calcified soft tumors including three cases of thyroid papillary carcinoma, three of breast invasive ductal carcinoma, three of prostate cancer, three of colon adenocarcincoma and three of normal lymph nodes, were selected in this study, and so were nine cases of bone and bone marrow tissue from biopsy and autopsy. Tissue samples were divided into three groups. One group was each soaked into 10% nitric acid for 24, 42 and 48 h, another group was each incubated in 1% hydrochloric acid for 1, 2 and 3 h. The other group which was not treated with acid was regarded as control. Then immunohistochemical staining was conducted in these samples for antigens as follows: ER, PR and Ki-67 in breast invasive ductal carcinoma; TTF-1 in thyroid papillary carcinoma; 34βE12 and P504s in prostate cancer; AE1/AE3, P504s and Ki-67 in colon adenocarcincoma; CD3, CD20 and Ki-67 in lymph nodes; CD3,CD20,CD68,CD235a,MPO,Ki-67,AE1/AE3 and TTF-1 in bone tissue., Results: After the tissues decalcificated in both acid solutions for some time, the numbers of positive cells decreased and the intensity of immunostaining became weaker, which was proportionate to time. Bone tissue was less influenced than calcified soft tissue. Different antigens showed different sensitivity to the same acid treatment. TTF-1 and Ki-67 were the two most vulnerable antigens among them; while ER, CD3 and CD20 were the least influenced. The effects of decalcification on immunostaining of one antigen were the same in different tissues., Conclusion: In most cases, both hydrochloric acid and nitric acid decalcification can lower the sensitivity of immunohistochemical staining, but have different effects on different antigens. It is better to use 10% nitric acid for decalcification of relatively large bone tissue and to use 1% hydrochloric acid for bone marrow tissues and bone tissues containing much more soft tissue.

Immunohistochemistry of undecalcified bone sections embedded in methyl methacrylate (MMA) is not commonly employed because of potential destruction of tissue antigenicity by highly exothermic polymerization. The aim of the present study was to describe a new technique in which a quick decalcification of bone sections embedded in MMA improves the results for immunohistochemistry. The quality of interleukin 1alpha (IL-1alpha) immunostaining according to the present method was better than the conventional one. Immunostaining for osteoprotegerin (OPG) and the receptor activator of NF-kappaB ligand (RANKL) in bone sections of chronic kidney disease patients with mineral bone disorders (CKD-MBD) was stronger than in controls (postmortem healthy subjects). The present study suggested that this method is easy, fast, and effective to perform both histomorphometry and immunohistochemistry in the same bone fragment, yielding new insights into pathophysiological aspects and therapeutic approaches in bone disease.