Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology.

The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.

Immunostaining of Skeletal Tissues.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic and translational research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with mineralized tissues is more challenging than with soft tissues. Demineralizing the samples allows for embedding in paraffin wax, and also facilitates cryosectioning. This chapter describes methods for IHC on formaldehyde-fixed, demineralized, paraffin-embedded, or frozen sections to detect antigens in skeletal tissues.

Double Immunohistochemistry and Digital Image Analysis.

Immunohistochemistry (IHC) is a commonly used technique for protein detection in tissue sections. The method requires high-affinity antibodies that are specific for the target proteins of interest. More advanced IHC techniques have been developed to meet the need for simultaneous detection of more than one target protein in the same tissue section. This chapter provides general guidelines for double IHC staining of formalin-fixed, paraffin-embedded tissue sections. Chromogenic substrates are chosen based on their excellent contrast and compatibility with the subsequent digital image analysis.

Multispectral Fluorescence Imaging Allows for Distinctive Topographic Assessment and Subclassification of Tumor-Infiltrating and Surrounding Immune Cells.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.

Paraffin-embedding lithography and micro-dissected tissue micro-arrays: tools for biological and pharmacological analysis of ex vivo solid tumors.

There is an urgent need and strong clinical and pharmaceutical interest in developing assays that allow for the direct testing of therapeutic agents on primary tissues. Current technologies fail to provide the required sample longevity, throughput, and integration with standard clinically proven assays to make the approach viable. Here we report a microfluidic micro-histological platform that enables ex vivo culture of a large array of prostate and ovarian cancer micro-dissected tissue (MDT) followed by direct on-chip fixation and paraffination, a process we term paraffin-embedding lithography (PEL). The result is a high density MDT-Micro Array (MDTMA) compatible with standard clinical histopathology that can be used to analyse ex vivo tumor response or resistance to therapeutic agents. The cellular morphology and tissue architecture are preserved in MDTs throughout the 15 day culture period. We also demonstrate how this methodology can be used to study molecular pathways involved in cancer by performing in-depth characterization of biological and pharmacological mechanisms such as p65 nuclear translocation via TNF stimuli, and to predict the treatment outcome in the clinic via MDT response to taxane-based therapies.

Single-Cell Mass Cytometry of Archived Human Epithelial Tissue for Decoding Cancer Signaling Pathways.

The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.

Morphological description of male genital organs of Marca's marmoset (Mico marcai).

Morphological characterisation of the genital organs of primates may bring significant contributions to the understanding of different reproductive behaviours and support new conservation strategies. However, relevant or detailed descriptions of genital morphology of several primate species are still lacking. This study describes the gross and microscopic anatomy of the internal and external genitalia of Marca's marmoset (Mico marcai). The same organs described in other primate species were identified here, but some anatomical particularities were detected, such as absence of a dartos tunic, presence of a vas deferens ampulla, absence of spongious erectile tissue in the pelvic urethra, separation of prostate gland lobes by a longitudinal sulcus and lack of septation in the corpus cavernosus and spongiosus at the level of the shaft and free portion of the penis. Keratinised type 1 spicules arising from epidermal or dermal projections were found in the free portion of the penis. Microscopic analysis revealed a small bone (baculum) consisting of peripheral compact bone and a central, non-ossified area filled with vascular tissue at the distal end of this portion of the penis. Results of this study may support further comparative studies of primates' reproductive ecology.

Preparation of Cells from Formalin-Fixed, Paraffin-Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments.

Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin-fixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 µm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50-µm tissue sections. To interpret FISH results using 4 to 6 µm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei from the single-cell suspension generally gives an interpretable result.

Preparation of formalin-fixed paraffin-embedded tissue for immunohistochemistry.

The purpose of this protocol is to take any biopsy or whole organ tissue from animals or human, formalin-fix the specimen to preserve the current state of the tissue, and embed it into a paraffin block and for future immunohistochemistry experiments (If you intend to fix cells, check the alternative protocols: Preparation of Cells for Microscopy using Cytospin, Preparation of Cells for Microscopy using Chamber Slides and Coverslips, or Preparation of Cells for Microscopy using 'Cell Blocks').

Preparation of cells for microscopy using 'cell blocks'.

Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Embedding cells in agarose and then in paraffin as 'cell blocks' allows for them to be processed in the same manner that tissue specimens are processed for histology. This method is advantageous because numerous sections can be cut from one cell block. Additionally, sectioning renders antigens within the nucleus and other cell organelles more accessible to antibodies. However, access to specialized equipment for histological tissue processing is required See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using Cytospin and for adherent cells on Preparation of Cells for Microscopy using Chamber Slides and Coverslips.

No-cost manual method for preparation of tissue microarrays having high quality comparable to semiautomated methods.

Manual tissue microarray (TMA) construction had been introduced to avoid the high cost of automated and semiautomated techniques. The cheapest and simplest technique for constructing manual TMA was that of using mechanical pencil tips. This study was carried out to modify this method, aiming to raise its quality to reach that of expensive ones. Some modifications were introduced to Shebl's technique. Two conventional mechanical pencil tips of different diameters were used to construct the recipient blocks. A source of mild heat was used, and blocks were incubated at 38°C overnight. With our modifications, 3 high-density TMA blocks were constructed. We successfully performed immunostaining without substantial tissue loss. Our modifications increased the number of cores per block and improved the stability of the cores within the paraffin block. This new, modified technique is a good alternative for expensive machines in many laboratories.

Evaluation of a completely automated tissue-sectioning machine for paraffin blocks.

Tissue sectioning automation can be a resourceful tool in processing anatomic pathology specimens. The advantages of an automated system compared with the traditional manual sectioning rely on the consistency of the final sectioned material translated into invariable thickness, uniform orientation during serial sectioning and less tissue sectioning artifacts. This technical note presents the design of an automated tissue-sectioning device and compares the sectioned specimens with normal manual tissue sectioning performed by experienced histology technician.

Avaliação de combinações de técnicas alternativas de construção e montagem de blocos de tissue microarray / Evaluation of alternative technique combinations of building and preparation of tissue microarrays blocks

INTRODUÇÃO E OBJETIVOS: O objetivo deste estudo foi pesquisar diferentes métodos alternativos de tissue microarray (TMA) à técnica original e conduzir adaptações desses, combinando diferentes métodos de punção das amostras teciduais e de montagem dos blocos de TMA, de modo a introduzir no Laboratório de Patologia Bucal da Faculdade de Odontologia de Pernambuco da Universidade de Pernambuco (LPBFOP/UPE) técnicas de TMA facilmente operáveis, reproduzíveis e de baixo custo. RESULTADOS: Foram reproduzidas quatro técnicas de punção dos blocos doadores e duas de montagem dos blocos de TMA, resultando em oito combinações possíveis. Para cada combinação, foram confeccionados três blocos de TMA, contendo nove, 16 e 32 amostras, respectivamente, e avaliadas quanto a perda de amostras, custo, tempo de confecção e dificuldade. Para blocos com nove amostras, a combinação 2 mostrou-se a mais adequada; para blocos com 16, a combinação 6 foi constatada como a mais eficiente; e para blocos com 32, a combinação 1 apresentou o melhor custo-benefício. CONCLUSÃO: Foi concluído que a escolha da combinação a ser utilizada depende do número de amostras a serem colocadas nos blocos de TMA.

INTRODUCTION AND OBJECTIVES: The aim of this study was to investigate different alternative tissue microarray (TMA) techniques and to make adaptations, combining different tissue punch and TMA block construction techniques in order to introduce easily reproducible, operational and cost effective TMA techniques in the Oral Pathology Laboratory of Pernambuco College of Dentistry, State University of Pernambuco. METHODS: Four donor punch techniques and two TMA block construction techniques were performed, resulting in a total of eight possible combinations. For each combination three TMA blocks were made, containing 9, 16 and 32 samples, respectively. They were evaluated as to sample loss, cost effectiveness, construction time and difficulty. RESULTS: For blocks with 9 samples, combination 2 was the most appropriate; for blocks with 16, combination 6 was the most efficient; and for blocks with 32, combination 1 was the most cost effective. CONCLUSION: It was concluded that the combination choice depends on the number of samples to be put in TMA blocks.

Transfer and multiplex immunoblotting of a paraffin embedded tissue.

In the functional proteome era, the proteomic profiling of clinicopathologic annotated tissues is an essential step for mining and evaluations of candidate biomarkers for disease. Previously, application of routine proteomic methodologies to clinical tissue specimens has provided unsatisfactory results. Multiplex tissue immunoblotting is a method of transferring proteins from a formalin-fixed, paraffin-embedded tissue section to a stack of membranes which can be applied to a conventional immunoblotting method. A single tissue section can be transferred to up to ten membranes, each of which is probed with antibodies and detected with fluorescent tags. By this approach, total protein and target signals can be simultaneously determined on each membrane; hence each antibody is internally normalized. Phosphorylation-specific antibodies as well as antibodies that do not readily work well with paraffin-embedded tissue are applicable to the membranes, expanding the menu of antibodies that can be utilized with formalin-fixed tissue. This novel platform can provide quantitative detection retaining histomorphologic detail in clinical samples and has great potential to facilitate discovery and development of new diagnostic assays and therapeutic agents.

One-step complete melting of paraffin tissue microarrays using stabilization bodies.

Paraffin tissue microarrays (PTMAs) are constructed by putting paraffin tissue core biopsies (PTCBs) from donor blocks into the preformed holes of a recipient block. One problem that can occur during sectioning of a PTMA is that PTCBs can fold up. This may be caused by insufficient adhesion of the paraffins of the PTCBs and the recipient block. Mengel et al solved this problem by melting the PTMA block in a 2-step melting procedure. To simplify this melting procedure by reducing it to 1 step, we propose the use of paraffinized stabilization bodies, that is tissue or other biologic or synthetic material, for the construction of PTMAs. Because they keep their structure while the PTMA melts, they stabilize the PTCBs. Thus, a PTMA with a stabilization body can be fully melted in 1 step with a routine device like a hot plate without danger of the PTCBs toppling over.

High-throughput zebrafish histology.

The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism's small larvae also makes it a potentially important model for toxicological analysis. Studying histological phenotypes is greatly enhanced by high-throughput methods of histology. Here, we describe details of high-throughput histology of the zebrafish using larval arrays, along with recent advances in mold design and discussion of work in progress that will lead to easier ways for people in the field to more rapidly score phenotypes in arrays. These detailed descriptions, together with the troubleshooting guide, should enable any laboratory with ties to a histology facility to perform high-throughput histology of zebrafish.

Three-dimensional reconstruction of tumor microvasculature: simultaneous visualization of multiple components in paraffin-embedded tissue.

Three-dimensional (3D) visualization of microscopic structures may provide useful information about the exact 3D configuration, and offers a useful tool to examine the spatial relationship between different components in tissues. A promising field for 3D investigation is the microvascular architecture in normal and pathological tissue, especially because pathological angiogenesis plays a key role in tumor growth and metastasis formation. This paper describes an improved method for 3D reconstruction of microvessels and other microscopic structures in transmitted light microscopy. Serial tissue sections were stained for the endothelial marker CD34 to highlight microvessels and corresponding images were selected and aligned. Alignment of stored images was further improved by automated non-rigid image registration, and automated segmentation of microvessels was performed. Using this technique, 3D reconstructions were produced of the vasculature of the normal brain. Also, to illustrate the complexity of tumor vasculature, 3D reconstructions of two brain tumors were performed: a hemangioblastoma and a glioblastoma multiforme. The possibility of multiple component visualization was shown in a 3D reconstruction of endothelium and pericytes of normal cerebellar cortex and a hemangioblastoma using alternate staining for CD34 and alpha-smooth muscle actin in serial sections, and of a GBM using immunohistochemical double staining. In conclusion, the described 3D reconstruction procedure provides a promising tool for simultaneous visualization of microscopic structures.