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Postharvest fibrosis and greening of Toona sinensis buds significantly affect their quality during storage. This study aimed to clarify the effects of low-temperature storage on postharvest red TSB quality harvested in different seasons. Red TSB samples were collected from Guizhou province, China, 21 days after the beginning of spring (Lichun), summer (Lixia), and autumn (Liqiu), and stored at 4 °C in dark conditions. We compared and analyzed the appearance, microstructure, chlorophyll and cellulose content, and expression levels of related genes across different seasons. The results indicated that TSB harvested in spring had a bright, purple-red color, whereas those harvested in summer and autumn were green. All samples lost water and darkened after 1 day of storage. Severe greening occurred in spring-harvested TSB within 3 days, a phenomenon not observed in summer and autumn samples. Microstructural analysis revealed that the cells in the palisade and spongy tissues of spring and autumn TSB settled closely during storage, while summer TSB cells remained loosely aligned. Xylem cells were smallest in spring-harvested TSB and largest in autumn. Prolonged storage led to thickening of the secondary cell walls and pith cell autolysis in the petioles, enlarging the cavity area. Chlorophyll content was higher in leaves than in petioles, while cellulose content was lower in petioles across all seasons. Both chlorophyll and cellulose content increased with storage time. Gene expression analysis showed season-dependent variations and significant increases in the expression of over half of the chlorophyll-related and cellulose-related genes during refrigeration, correlating with the observed changes in chlorophyll and cellulose content. This research provides valuable insights for improving postharvest storage and freshness preservation strategies for red TSB across different seasons.

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BACKGROUND: PD-L1 staining using long-stored paraffin sections may not be consistent with the true PD-L1 expression of patients. Therefore, it is necessary to explore the expression of PD-L1(SP142) in paraffin sections of invasive breast cancer with different storage times and the optimal storage temperature for unstained paraffin sections. METHODS: The study included 71 cases of PD-L1(SP142) positive breast cancer. The unstained paraffin sections were stored at room temperature conditions (20-25 °C), 4 °C, -20 °C and - 80 °C, respectively. PD-L1 staining was performed at 1, 2, 3, 4, 8, 12 and 24 weeks of storage. PD-L1 expression was assessed with a continuity score. RESULTS: The PD-L1 antigen was gradually lost as the storage time of paraffin sections increased. The PD-L1 positivity rate was 97.18% at 1 week for the sections stored at room temperature, and decreased from 83.10 to 71.83% for the sections stored for 2 weeks to 4 weeks, and 61.97%, 54.93%, and 32.93% for 8, 12, and 24 weeks, respectively. When stored at low temperatures of 4 °C, -20 °C and - 80 °C, the positivity rate decreases with the same trend but more slowly compared to room temperature. The mean IC score of PD-L1 also showed a gradual decrease in all cases. In the consistency analysis, PD-L1 expression in slices stored at room temperature for 2 weeks was consistent with PD-L1 expression in fresh slices (ICC ≥ 0.9 for all slices), and PD-L1 expression in slices stored at 4 °C or -20 °C for 4 weeks was consistent with PD-L1 expression in fresh slices (ICC ≥ 0.9 for all slices). When stored under refrigeration at -80 °C, PD-L1 expression in slices stored for 3 weeks was consistent with that in fresh slices (ICC ≥ 0.9). CONCLUSIONS: To our knowledge, this is the first article on the effect of preservation time and preservation temperature of paraffin sections on PD-L1 expression in breast cancer. Long-term storage of paraffin sections of unstained invasive breast cancer can lead to antigen loss of PD-L1 (SP142). Refrigerated storage of paraffin sections can delay antigen loss, with best results at 4 °C or -20 °C, and a storage time of no more than 4 weeks is recommended.

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Planarians are a model animal for the study of regeneration and homeostasis. Understanding how planarians control their cellular balance is key to the knowledge of their plasticity. Both apoptotic and mitotic rates can be quantified in "whole mount" planarians. Apoptosis is usually analyzed through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), a technique that detects cell death by identifying DNA breaks. In this chapter we detail a protocol to analyze apoptotic cells in paraffin sections of planarians, which enables a more accurate cellular visualization and quantification than in "whole mount."

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There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during active inflammatory bowel disease (IBD), which is associated with an increased influx of immune cells into the gut mucosa. Therefore, studies on how chemokines are regulated in the intestinal epithelium may identify putative treatment targets in IBD. More recently, patient-derived ex vivo models such as intestinal organoids have facilitated molecular analysis of epithelial alterations in IBD patients own cells. Here, we describe refined experimental protocols and methods for the generation and maintenance of IBD patient-derived colonic organoids (colonoids) culture. We also give detailed description of medium, and supplements needed for colonoid establishment, growth, and differentiation, including production of Wnt-3A and Rspondin1 enriched media. Further, we present protocols for RNA and protein isolation from human colonoids, and subsequent gene expression analysis and Western blotting for e.g., signal transduction studies. We also describe how to process colonoids for chemokine protein expression analysis such as immunostaining, confocal imaging, and detection of secreted chemokines by e.g., enzyme-linked immunosorbent assay (ELISA). As proof of principle, we give examples of how the chemoattractant CCL20 can be regulated and expressed in colonoids derived from IBD-patients and healthy controls upon ligands-driven inflammation.

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The function of glycoproteins depends both on their polypeptide chain and sugar residues. For detection and localization of glycoproteins in tissue sections, methods of immunohistochemistry (IHC) and lectin histochemistry (LHC) are usually used separately. For a better understanding of the expression and distribution of variants of glycoproteins, tissue sections can be analyzed by combined lectin- and immuno-histochemistry (CLIH). CLIH exploits the advantages of both IHC and LHC and can therefore contribute to research in glycobiology and other fields of cell biology. Since cancer transformation is accompanied by alterations in the glycosylation of some glycoproteins, CLIH could also be exploited for improved classification of cancers. The chapter considers how CLIH could be employed on paraffin sections and semithin cryosections for fluorescence microscopy. Five different protocols of CLIH are described in detail as well as appropriate negative controls.

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Formalin fixed paraffin embedded tissue occasionally requires reprocessing if the histologic quality of a section is inadequate for clinical diagnosis. The Pat Dry (PD) and the Serial Xylene (SX) methods are two techniques described in the literature to reprocess under-fixed and/or under-processed tissue samples. To date, no study has compared the effects of these methods on the histologic quality of tissue sections, cost, and turnaround times. In the present study, these two methods were evaluated on 129 tissue samples taken from 40 submitted clinical specimens, 3 blocks per sampled location. Before processing, sample Group 1 (Control) was cut at routine 3-5 mm thickness. Sample Groups 2 and 3 were cut at 10 mm to ensure the thicker tissues would be poorly processed. Histotechnicians performed a subjective evaluation of all the samples at the time of embedding and microtomy. Hematoxylin and eosin stained sections from all samples were scored for histologic quality by two pathology residents. Thicker samples (Groups 2 and 3) were then reprocessed using either PD or SX methods, re-sectioned, stained, and then re-scored by the pathology residents. The two reprocessing methods equally improved quality scores and reduced the fraction of slides that were rejected. The PD method average preparation time was 66 minutes as compared to 250 minutes for the SX method. The PD method was easier to perform than the SX method, required less reagent, and was less susceptible to reagent spillage than the SX method.

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BACKGROUND: The fast acquisition process of frozen sections allows surgeons to wait for histological findings during the interventions to base intrasurgical decisions on the outcome of the histology. Compared with paraffin sections, however, the quality of frozen sections is often strongly reduced, leading to a lower diagnostic accuracy. Deep neural networks are capable of modifying specific characteristics of digital histological images. Particularly, generative adversarial networks proved to be effective tools to learn about translation between two modalities, based on two unconnected data sets only. The positive effects of such deep learning-based image optimization on computer-aided diagnosis have already been shown. However, since fully automated diagnosis is controversial, the application of enhanced images for visual clinical assessment is currently probably of even higher relevance. METHODS: Three different deep learning-based generative adversarial networks were investigated. The methods were used to translate frozen sections into virtual paraffin sections. Overall, 40 frozen sections were processed. For training, 40 further paraffin sections were available. We investigated how pathologists assess the quality of the different image translation approaches and whether experts are able to distinguish between virtual and real digital pathology. RESULTS: Pathologists' detection accuracy of virtual paraffin sections (from pairs consisting of a frozen and a paraffin section) was between 0.62 and 0.97. Overall, in 59% of images, the virtual section was assessed as more appropriate for a diagnosis. In 53% of images, the deep learning approach was preferred to conventional stain normalization (SN). CONCLUSION: Overall, expert assessment indicated slightly improved visual properties of converted images and a high similarity to real paraffin sections. The observed high variability showed clear differences in personal preferences.

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Immunofluorescence staining is the most frequently applied technique to detect and visualize various molecules in biological samples. Many protocols can be found in the literature and the websites of commercial antibody producers. This can result in a time-consuming and costly methodical work to establish "simple" antibody staining. We here summarize in a stepwise manner an easy-to-follow immunofluorescence staining protocol with an improved specific fluorescent signal and a reduced background and non-specific binding signal. This will help scientists to save time, effort, and antibody costs during the application of such a valuable technique.

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Either through differentiated glands or specialised individual cells, the coating epithelia of soft-bodied marine invertebrates are responsible for the secretion of a broad span of peptidic substances, from protective mucins to biocides. These secretions are characterised by the presence of cysteine-rich proteins and peptides, rendering a distinct histochemical signature of secretory epithelia. Through a histochemical procedure for fluorescence microscopy in paraffin sections, we performed a comparative assessment of the distribution of thiol-rich compounds in multiple epithelia of different species of intertidal Polychaeta, which revealed distinctive patterns of distribution that closely relate to ecology, morphoanatomy and physiology. The presence of free thiols was notorious in mucocytes and enzyme-plus toxin-secreting cells. Consequently, strong signals were recorded in the mucocytes of the parapodia of Nereis splendida, the epidermis and pharynx epithelium of Mysta picta and the venom glands of Glycera alba. The findings show an investment in mucus secretion in foragers such as Nereis and Mysta, especially the latter, which is not a native burrower, as a protective response and as lubricant for locomotion. Additionally, nereidids are believed to secret integumentary toxins for defence. On the other hand, Glycera is an ambush predatorial burrower whose behaviour entirely revolves around the delivery of venom making use of its four jaws. The results showed that the detection of thiol-rich compounds in histological sections can be a tool to identify potential toxin secretion and delivery structures, with important consequences for the bioprospecting of novel bioreactives from marine invertebrates for the purpose of drug discovery.

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INTRODUCTION: There are few published studies comparing immunofluorescence on formalin-fixed, paraffin-embedded (FFPE) tissue sections (IF-P) and immunoperoxidase on FFPE tissue sections (IP-P) with immunofluorescence on frozen sections (IF-F) for evaluation of renal diseases. Also, the accuracy for each method differs greatly. The aim of this study was to evaluate IF-P and IP-P as an alternative to IF-F (gold standard method) in the diagnosis of renal biopsies specimens. METHODS: In all, 101 renal biopsies were subjected to IF-P, IP-P, and IF-F staining to demonstrate immunoglobulin IgA, IgG, and IgM immune deposits. Sensitivity, specificity, false-positive, and false-negative values were calculated. RESULTS: IP-P showed sensitivity of 61.8%, 74.2%, and 64.2%, and specificity of 84.8%, 69.2%, and 66.7% for IgA, IgG, and IgM, respectively. IF-P showed a sensitivity of 45.6%, 69.4% and 52.8%, and specificity of 87.9%, 87.2% and 77.1% for IgA, IgG and IgM, respectively. False-positive cases of IF-P and IP-P were 4, 5, and 11 and 5, 12, and 16 for IgA, IgG, and IgM, respectively. CONCLUSION: Where IF-F lacks glomeruli or fresh renal biopsies are not available, IP-P is a sensitive method, whereas IF-P is a specific method for the evaluation of immune deposits in the renal tissue biopsies. The presence of false-positive cases in both methods deserves further research.

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Fluorescence in situ hybridization (FISH) is a specific, sensitive, accurate, and reliable technique widely applied in both research and clinic. Here we describe the detailed protocol of a FISH method established by us to serve the scientific purposes of the first oncolytic parvovirus clinical trial (ParvOryx01). This trial was launched in Germany in 2011. After trial completion in 2015, results were published in Molecular Therapy in 2017. The primary purpose of the trial was to evaluate the safety of an oncolytic parvovirus, H-1PV (ParvOryx), in recurrent glioblastoma patients. In addition, the efficiency of H-1PV tumor targeting after intratumoral or systemic virus administration was assessed by FISH detection of viral nucleic acids (genomic single-stranded DNA, mRNA and parvovirus double-stranded replicative forms) in formalin-fixed paraffin-embedded glioblastoma tissues resected at day 10 after ParvOryx treatment. The FISH method allowed the detection-for the first time in humans-of H-1PV replication markers in brain tumors of parvovirus-treated patients. A protocol combining mRNA FISH with simultaneous immunofluorescent staining for tumor and tumor microenvironment markers was also developed and is described here, in order to better characterize H-1PV cellular targets and H-1PV treatment-associated tumor microenvironment changes.

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The optical disector, a three-dimensional counting frame or probe in stereology, is often positioned in the middle (depth) of a thick section for unbiased nuclear counting. Using 30-40 µm thick methacrylate or paraffin sections for nuclear counting of neurons with the optical disector, however, some studies showed markedly higher nuclear densities at 10% of the section thickness near the top or bottom surface of the section, suggestive of deformation of section along its z axis and thus affecting the number estimation. To verify the findings, this study obtained two sets of 12-14 methacrylate sections (average thicknesses 21.7 and 29.4 µm) and two sets of 12 paraffin sections (average thicknesses 13.8 and 29.2 µm) from mature rat testes. Each section was used to count round spermatid nuclei in the seminiferous epithelium densely packed with the cells, using 3-4 consecutive disectors placed vertically (along the z axis of the section) from the top surface of the section, through the whole section thickness (two sets of methacrylate and paraffin sections) or in 80-83% of the thickness (other sections). The results demonstrated that, overall, there were no considerable nonuniform changes of the nuclear densities along the z axis of the sections.

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AIM: Tissue shrinking due to fixation and processing is well known. However, the degree of shrinking varies significantly with the tissue type as well as the processing method and is not well studied in various tissues. In daily pathological routine workflow, histological specimens from frozen and paraffin sections are performed from the same tissue. In the present study we compared the thickness of bronchus walls obtained from paraffin and frozen sections. METHODS: Pig lungs were frozen in ventilated condition in liquid nitrogen and 36 bronchi were isolated after dissection. Frozen sections of 5 µm thickness were performed and the remaining tissue was fixed and embedded in paraffin after fixation in 4% formalin. Frozen and paraffin sections from the same cutting edge were analysed after haematoxylin and eosin staining by measuring the wall thickness of the bronchi using high power fields of 400-fold magnification. In each bronchus 40 measurements were implemented at different wall positions distributed over the entire wall area. Summed up, in each group 1440 wall measurements were performed in total. Statistical analysis was conducted using the Wilcoxon test and t-test as well as Pearson's correlation coefficient with a significance level at P < 0.05. RESULTS: The bronchial wall thickness was significantly (p < 0.001) smaller in frozen sections (median: 0.50 mm; min: 0.37 mm; max: 0.97 mm) compared to paraffin sections (median: 0.58 mm; min: 0.35 mm; max: 1.06 mm). The median difference between paraffin and frozen sections was 0.05 mm (min: -0.11 mm; max: 0.22 mm). The wall thickness ratio of both groups was as follows: frozen/paraffin section = 0.8609, thus yielding a difference between paraffin and frozen of 13.91%. High correlation was found between wall thickness measurements on paraffin and frozen sections (R = 0.87, p < 0.001). CONCLUSIONS: The bronchus wall thickness in the frozen section was 14% reduced compared to the paraffin section. In routine pathology as well as in scientific studies these results are of relevance, as airway wall thickness represents a relevant marker for pathological interpretation, especially using CT image techniques.

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Immunohistochemistry (IHC) is a technique based on the specificity of antibody-antigen principle used commonly to detect antigens in tissue sections. The immune labeling can be performed in paraffin sections, cryostat sections, and ultrathin sections and can be observed in light confocal and transmission electron microscopy. However, the use of immunohistochemical techniques for the study of mineralized tissues has been a challenge for decades (Berdal et al., Arch Oral Biol 36:715-725, 1991; Nanci et al., Eur J Histochem 52:201-214, 2008). Specific procedures are necessary when compared with soft tissue immunohistochemistry. This chapter describes methods for IHC on Tissue-Tek O.C.T. compound and paraffin-embedded sections to detect antigens in the dental mineralized tissues.

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Formalin-fixed, paraffin-embedded tissue (FFPE) still plays an important role in biobanking, since it is comparatively easy to obtain and store in comparison to fresh frozen tissue. They are stored as paraffin or FFPE blocks. Unstained slides derived from FFPE blocks may be used for hematoxylin and eosin histology, special stains, immunohistochemistry, and chromogenic or fluorescent in situ hybridization. In addition, tissue scraped off FFPE slides or from scrolls of FFPE tissue may be used for molecular or proteomic analyses. Hematoxylin and eosin staining of FFPE sections reviewed by a pathologist are highly valuable to ensure the presence of adequate lesional cells for molecular and other analyses. Therefore, proper microtomy technique is essential in the preparation of formalin-fixed, paraffin-embedded tissue for biobanking purposes. Here we describe the process of cutting paraffin embedded sections using a rotary microtome. We also highlight the possible pitfalls that may arise and discuss how to avoid them.

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Lymph node diagnostics are essentially based on cutting thin sections of formalin fixed tissues. After hematoxylin and eosin stain, Giemsa stain and immunohistochemical staining of these tissues, the lymph node diagnosis is done using a light microscope, looking at two-dimensional pictures. Three-dimensional visualizations of lymph node tissue have not been used in lymphoma diagnostics yet. This article describes three-dimensional visualization of lymphoid tissue, using thick paraffin sections, immunostained with monoclonal antibodies, confocal laser scanning and data processing with appropriate software and the 3D printing process itself. The advantages and disadvantages of different printing techniques are discussed as well as the application of 3D models in diagnostics, teaching and research of lymph nodes.

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RNA in situ hybridization techniques are an important tool for the study of gene expression patterns in freshwater planarians. Here I describe a RNA in situ hybridization method on histological paraffin sections of planarian tissue. This protocol allows the visualization of gene expression at cellular or subcellular resolution. Following paraffin-embedding and sectioning of planarians, the resulting sections are hybridized with hapten-labeled RNA probes. Subsequent immunological probe detection is carried out with either chromogenic or fluorescent development. This protocol can be performed alone, or in combination with other immunodetection techniques, and represents a useful alternative to whole-mount protocols more commonly used in the community.

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The concerns about the presence of microplastics (MPs) in marine ecosystems have widely increased in the past years. This is reflected in a growing number of studies addressing the effects of exposure to these materials in indigenous, farmed and even laboratory marine animals subjected to toxicity-oriented bioassays. There have been, however, many constraints in the detection of MPs in biological tissues, as routine histological techniques tend to degrade these materials, which are especially sensitive to organic solvents. This issue hinders the application of standard histopathological procedures based on convenient paraffin wax-embedding protocols, with consequences for biomonitoring and bioassay procedures. The method described here was developed and validated for the detection of polystyrene microplastics in biological tissue processed for paraffin-based histology. The strategy was developed and tested from whole-soft body sections of marine mussels that internalised the MPs following dedicated bioassays. The protocol is based on the replacement of xylenes with isopropanol for the purpose of intermediate infiltration and deparaffinization. Special modifications for staining, mounting and archiving are needed and are detailed as well. The protocol is shown to be a highly cost- and time-effective procedure compatible with formalin-based fixatives plus standard sectioning and staining, yielding complete preservation of MPs and optimal tissue conditioning. The method also produced excellent results with pre-stained MPs, with fluorochromes included, altogether providing excellent localisation of polystyrene MPs in paraffin-processed biological tissue.

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Most human and animal biopsy samples are routinely embedded in paraffin since this enables the pathologist or researcher to obtain excellent morphology and simplifies storage. Nevertheless, in many cases, the antigen of interest cannot be detected in paraffin section. The alternative available for good immunohistochemistry is preparation of cryosections, which usually provide decent antigen preservation and are frequently used for immunofluorescence. However, cryosections often do not provide efficient morphological details of tissues and cells for pathologic evaluation. In order to obtain good antigen preservation and improve tissue and cell morphology after freezing, we tested three different fixations and freezing methodologies and compared them to routine formaldehyde fixation and paraffin embedding. As a model system, we selected the epithelium of the rat urinary bladder and trachea. On all samples, haematoxylin and eosin staining was performed as well as immunofluorescence with antibodies against tight junction protein ZO-1 and against intermediate filament cytokeratin 7. The best compromise between morphology and immunofluorescence was obtained with "sucrose impregnation prior to freezing" method. Moreover, this procedure is also quicker in comparison to standard paraffin section preparation. To check the clinical relevance of our study, this method was used for human biopsy samples of neoplastic urothelial and bronchial mucosa lesions. Besides good immunofluorescence results, the morphology of these samples was well preserved. We therefore propose that cryosection preparation with sucrose impregnation prior to freezing should be further exploited in other clinical and veterinary applications, since it enables good morphology and antigen preservation.

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Immunohistochemistry (IHC) is an efficient technique to detect cellular localizations of the proteins in paraffin-embedded tissues. It allows specific proteins to be visualized by the interaction of antibodies with an enzyme-substrate-chromogen system. Here, we describe indirect immunohistochemistry method for paraffin-embedded mouse ovaries fixed with Bouin's Fixative.

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