Western blotting (WB) and immunohistochemical staining (IHC) are common techniques for determining tissue protein expression. Both techniques require a primary antibody specific for the protein in question. WB data is band(s) on a membrane while IHC result is a staining on a tissue section. Most human genes are known to produce multiple protein isoforms; in agreement with that, multiple bands are often found on the WB membrane. However, a common but unspoken practice in WB is to cut away the extra band(s) and present for publication only the band of interest, which implies to the readers that only one form of protein is expressed and thus the data interpretation is straightforward. Similarly, few IHC studies discuss whether the antibody used is isoform-specific and whether the positive staining is derived from only one isoform. Currently, there is no reliable technique to determine the isoform-specificity of an antibody, especially for IHC. Therefore, cutting away extra band(s) on the membrane usually is a form of misconduct in WB, and a positive staining in IHC only indicates the presence of protein product(s) of the to-be-interrogated gene, and not necessarily the presence of the isoform of interest. We suggest that data of WB and IHC involving only one antibody should not be published and that relevant reports should discuss whether there may be protein multiplicity and whether the antibody used is isoform-specific. Hopefully, techniques will soon emerge that allow determination of not only the presence of protein products of genes but also the isoforms expressed.

MicroRNA (miRNA) is a class of small non-coding RNAs which mediate post-transcriptional gene silencing (PTGS) by sequence-specific inhibition of target mRNAs translation and/or lowering their half-lives in the cytoplasm. Together with their binding partners, Argonaute (AGO) proteins, miRNAs form cores of RNA-induced silencing complexes (RISC). Despite a substantial progress in understanding RISC structure, until recently little was known about its localization in the cell. This review is aimed to provide an overview of the emerging picture of miRNA and RISC localization and function both in the intracellular space and outside of the cell. In contrast to the common assumption that PTGS occurs in the cytoplasm, it was found to operate mainly on the membranes of the endoplasmic reticulum (ER). Besides ER membranes miRNAs were found in all main cellular compartments including nucleus, nucleolus and mitochondria where they regulate various processes including transcription, translation, alternative splicing and DNA repair. Moreover, a certain pool of miRNAs may not be associated with RISC and carry completely different functions. Finally, the discovery of cell-free miRNAs in all biological fluids suggests that miRNAs might also act as signaling molecules outside the cell, and may be utilized as biomarkers for a variety of diseases. In this review we discuss miRNA secretion mechanisms and possible pathways of cell-cell communication via miRNA-containing exosomes in vivo.

L1CAM is a cell adhesion molecule of the immunoglobulin superfamily which was originally discovered as a major player in the development of the nervous system. L1CAM was demonstrated to have prognostic value in different cancers and to be a promising target for anti-cancer therapy. Here we overview the present data on L1CAM structure and function, regulation of its expression, role in cancer and therapeutic potential.

For 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular matrix such as dermis in skin or gingiva are difficult to clear. The present survey lists methodologies that are available for clearing of tissues for 3D imaging. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing connective tissue-rich gingiva and dermis of skin for 3D histochemistry and imaging of fluorescence using light-sheet microscopy.

This review discussed the importance of mutated tau, amyloid and neuroinflammatory factors and microglia in Alzheimer disease. In particular tau, CD4 and TNF alpha were included in the review and the colocalizations of these factors were highlighted. It is important to realize the Alzheimer disease may result from the interactions of these factors. Some of these factors may coexist at the same region and at the same time e.g. mutated tau and amyloid in plaques. A summary scheme of etiology leading to the disease was included.

In situ hybridisation (ISH) is unique amongst molecular analysis methods in providing for the precise microscopic localisation of genes, mRNA and microRNA in metaphase spreads, cell and tissue preparations. The method is well established as a tool to guide appropriate therapeutic intervention in breast, gastric and lung cancer. With the description of ultrasensitive ISH technologies for low copy mRNA demonstration and the relative ease by which microRNA can be visualised, the applications for research and diagnostic purposes is set to increase dramatically. In this review ISH is considered with emphasis on recent technological developments and surveyed for present and future applications in the context of the demonstration of genes, mRNA and microRNA in health and disease.

Articular cartilage guarantees for an optimal functioning of diarthrodial joints by providing a gliding surface for smooth articulation, weight distribution, and shock absorbing while the subchondral bone plays a crucial role in its biomechanical and nutritive support. Both tissues together form the osteochondral unit. The structural assessment of the osteochondral unit is now considered the key standard procedure for evaluating articular cartilage repair in translational animal models. The aim of this review is to give a detailed overview of the different methods for a comprehensive evaluation of osteochondral repair. The main focus is on the histological assessment as the gold standard, together with immunohistochemistry, and polarized light microscopy. Additionally, standards of macroscopic, non-destructive imaging such as high resolution MRI and micro-CT, biochemical, and molecular biological evaluations are addressed. Potential pitfalls of analysis are outlined. A second focus is to suggest recommendations for osteochondral evaluation.

Mas-related genes (Mrgs) belong to a large family of G protein-coupled receptor genes found in rodents. Human MRGX proteins are G protein-coupled 7-transmembrane proteins sharing 41-52% amino acid identity with each other, but have no orthologs in rodents. MrgX2 is a member of the MrgX family. MRGX2 is expressed in the small neurons of sensory ganglia and mast cells. It can interact with a series of factors and genes such as the peptides substance P, vasoactive intestinal peptide, cortistatin (CST), proadrenomedullin N-terminal peptide (PAMP), LL-37, PMX-53 and β-defensins. MRGX2 is related to nociception, adrenal gland secretion and mast cell degranulation. Recent research on MrgX2 provides insights into its role in nociception and anti-microbial activities. This article reviewed the origin, expression and function of MrgX2, and discussed possible future research focus.

Low voltage transmission electron microscopy (LVTEM) was employed to examine biological tissues with accelerating voltages as low as 5kV. Tissue preparation was modified to take advantage of the low-voltage techniques. Treatments with heavy metals, such as post-fixation with osmium tetroxide, on block and counterstaining were omitted. Sections (40nm) were thinner than usual and generated highly contrasted images. General appearance of the cells remains similar to that of conventional TEM. New features were however revealed. The matrix of the pancreatic granules displays heterogeneity with partitions that may correspond to the inner-segregation of their secretory proteins. Mitochondria revealed the presence of the ATP synthase granules along their cristea. The nuclear dense chromatin displayed a honeycomb organization while distinct beads, nucleosomes, aligned along thin threads were seen in the dispersed chromatin. Nuclear pore protein complexes revealed their globular nature. The intercalated disks in cardiac muscle displayed their fine structural organization. These features correlate well with data described or predicted by cell and molecular biology. These new aspects are not revealed when thicker and conventionally osmicated tissue sections were examined by LVTEM, indicating that major masking effects are associated with standard TEM techniques. Immunogold was adapted to LVTEM further enhancing its potential in cell biology.

The metastatic cascade comprises the following steps in sequential manner: the future metastatic cell has to leave the primary tumor mass, degrade the surrounding extracellular matrix, extravasate and circulate within in the bloodstream. Thereafter it has to attach to the endothelium of a target organ, intravasate into the connective tissue and has to proliferate to form a clinically detectable metastasis. We overview the in vitro microfluidic platforms modelling the metastatic cascade and the evolution towards systems capable of recapitulating all the steps by a single comprehensive model.