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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.

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.

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.

Bone metastasis from primary cancer sites creates diagnostic and therapeutic challenges. Calcified bone is difficult to biopsy due to tissue hardness and patient discomfort, thus limiting the frequency and availability of bone/bone marrow biopsy material for molecular profiling. In addition, bony tissue must be demineralized (decalcified) prior to histomorphologic analysis. Decalcification processes rely on three main principles: (a) solubility of calcium salts in an acid, such as formic or nitric acid; (b) calcium chelation with ethylenediaminetetraacetic acid (EDTA); or (c) ion-exchange resins in a weak acid. A major roadblock in molecular profiling of bony tissue has been the lack of a suitable demineralization process that preserves histomorphology of calcified and soft tissue elements while also preserving phosphoproteins and nucleic acids. In this chapter, we describe general issues relevant to specimen collection and preservation of osseous tissue for molecular profiling. We provide two protocols: (a) one-step preservation of tissue histomorphology and proteins and posttranslational modifications, with simultaneous decalcification of bony tissue, and (b) ethanol-based tissue processing for TheraLin-fixed bony tissue.

This study was performed to elucidate the relationship between dental caries and the levels of Sr and Ca eluted from enamel, and to examine whether these elements are useful as factors to assess caries risk. The available 103 (Sr) and 108 (Ca) samples were obtained among 111 collected deciduous teeth. The healthy regions of enamel were decalcified in artificial saliva at pH 6.2 and 5.5. The eluted levels of these elements from enamel were determined using atomic absorption spectrophotometry. Sr and Ca levels were not affected by the sex nor tooth type. Sr levels of the caries-experienced tooth (CE) group were 2.6-fold (pH 6.2) and 2.2-fold (pH 5.5) higher than those of the sound tooth (ST) group, respectively. Furthermore, the Sr levels were significantly higher in the teeth with treated than in those with untreated caries. Only at pH 6.2 was a significant difference found in Ca levels between the ST and CE groups. In the ST group, at pH 5.5, both the Sr and Ca levels significantly increased when the children had six or more carious teeth. The Sr and Ca elution levels were significantly inhibited in the teeth receiving fluoride application every 3 or 4 months compared to those that were not. These findings indicate that Sr can be an indicator of the acid resistance of teeth, and a useful factor to assess future caries risk.

Rodent arthritis models are tools to study joint destruction. This chapter describes methods to prepare and analyze histological sections from inflamed paws. Protocols to create decalcified paraffin-embedded as well as undecalcified plastic-embedded sections are provided. Strengths and limitations of these two histological techniques are described. Major staining techniques on hard tissue sections focusing on the evaluation of joint destruction are indicated. Finally, evaluation of arthritic paw sections by histomorphometry, a semiautomated computerized method to measure areas of interest, is described.