Understanding the interplay between commensals, pathogens, and immune cells in the skin and mucosal tissues is critical to improve prevention and treatment of a myriad of diseases. While ...high-parameter flow cytometry is the current gold standard for immune cell characterization in blood, it is less suitable for mucosal tissues, where structural and spatial information is lost during tissue disaggregation. Immunofluorescence overcomes this limitation, serving as an excellent alternative for studying immune cells in mucosal tissues. However, the use of immunofluorescent microscopy for analyzing clinical samples is hampered by a lack of high-throughput quantitative analysis techniques. In this chapter, we describe methods for sectioning, staining, and imaging whole sections of human foreskin tissue. We also describe methods to automate immune cell quantification from immunofluorescent images, including image preprocessing and methods to quantify both circular and irregularly shaped immune cells using open-source software.
Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic ...debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.
•The first immunofluorescence microscopy attempt of targeting two different types of proteins on the same cultural heritage micro sample.•A two-step antibody hybridization procedure to analyse two ...different types of protein binders in real wall paintings.•This procedure allowed us to reduce the costs and operational difficulties, which are normally encountered, when mixing two types of primary antibodies.•Ovalbumin was found in 3 micro samples and casein in 1 micro sample; and none proved positive for both binder types.
The aim of the study was to use immunofluorescence microscopy to identify two types of protein binders in 10 polished cross sections of micro samples, which were obtained from real and mock-up wall paintings. A two-step antibody hybridisation procedure was employed, in which the same micro samples were hybridised with anti-ovalbumin and subsequently with anti-casein antibodies and between the hybridisation steps, the sample's cross section was slightly re-polished and cleaned in order to remove all primary and secondary antibody remnants, which remained attached from the first hybridisation step (anti-ovalbumin). This allowed us to reduce the costs and operational difficulties, which are normally encountered, when antibodies, targeting two different proteins, are simultaneously mixed together. To reduce unspecific fluorescence, to amplify the fluorescence of the proteinaceous binders and to construct 3D surface topography models, apart from widefield fluorescence, laser-scanning confocal microscopy was performed. In parallel, FTIR spectroscopy analysis, of finding proteinaceous materials in micro sample cross sections, was also conducted. Results show that our two-step hybridisation procedure allowed for an accurate localisation of both types of proteinaceous binders without any interference from the first hybridisation step. Three micro samples proved positive for ovalbumin and one for casein and none of these proved positive for both analysed binder types. For eight micro samples, FTIR spectroscopy results completely matched those of immunofluorescence microscopy. According to our knowledge, this was the first immunofluorescence microscopy attempt of targeting two different types of proteins on the same cultural heritage micro sample.
•Can pigments within a paint hinder proteinous binder characterization using immunofluorescence microscopy?.•Eight out of 10 tested pigments did not interfere with binder characterization using ...immunofluorescence microscopy.•Five pigments exhibited weak autofluorescence, which did not interfere with immunofluorescence microscopy.•Confocal fluorescence gave clearer results to widefield fluorescence.•Cross sections with smooth surfaces gave a much clearer immunofluorescence microscopy picture.
The aim of the study was to address the problematics of proteinous binder characterization, within a cross section of painted model samples, using immunofluorescence microscopy. Problems arise from certain pigments which can alter the epitope sites of target binders (lead white and verdigris) or can exhibit a strong natural autofluorescence (lake pigments). Therefore, dual layered model samples were prepared containing a lower egg tempera paint and an upper oil paint, and both paints were made of the same pigment. As an extra challenge for fluorescence microscopy, half of samples were additionally covered with pure linseed oil (as a third layer), which is known to physically reflect fluorescence. Cross-sections were hybridized with anti-ovalbumin antibodies and with FITC labelled secondary antibodies. To reduce unspecific fluorescence, apart from widefield fluorescence, laser-scanning confocal immunofluorescence microscopy was performed. Finally, 3D surface topography models were constructed which were used to check off any unspecific fluorescence originating from cracks or holes. Results show that immunofluorescence microscopy in the widefield observation mode was successful in its specificity and clarity of highlighting the egg paint layer in the presence of 8 out of 10 pigments, including the problematic lead white and verdigris pigments. Several of these pigments (lead white, malachite, yellow ochre, madder lake and carbon black) exhibited autofluorescence; however it was not bright enough to interfere with the successful immunofluorescence microscopy result. In the widefield mode, immunofluorescence microscopy was unsuccessful in the presence of 2 pigments; carmine lake (pigment adsorbs antibodies) and Dragon's blood (pigment dissolved during resin curing at 50 °C). The confocal observation mode in comparison to the widefield mode achieved a much more specific and clear immunofluorescence microscopy picture (especially in the presence of Prussian blue, Vermilion and carbon black) and removed nearly all of the unspecific fluorescence originating from resin's surface reflection, from pure oil binder, from small indentations and from illumination glair that would have otherwise spread across different layers. Lastly, 3D topography models showed that in general samples with smooth surfaces gave a much clearer immunofluorescence microscopy picture.
•SEM experiments pinpointed that GF induced a significant aggregation of proteins.•Microscopy showed significant changes to gluten proteins after GF treatment.•Cross-reactivity with specific ...antibodies for gluten proteins was strongly reduced.
The main aim of this paper was to assess the impact of Gluten-Friendly™ (GF) technology (Italian priority patent n° 102015000084813 filed on 17th December 2015) on wheat kernel endosperm morphology and gluten protein structure, using SEM, light and immunofluorescent microscopy. Microscopy was combined with immunodetection with specific antibodies for gliadins, γ-gliadins, LMW subunits and antigenic epitopes to gain a better understanding of the technology at a molecular level. The results showed significant changes to gluten proteins after GF treatment; cross-reactivity towards the antibodies recognizing almost the entire range of gluten proteins as well as the antigenic epitopes through the sequences QQSF, QQSY, PEQPFPQGC and QQPFP was significantly reduced. The present study confirms the results from our previous work and shows, for the first time, the mechanism by which a chemical-physical treatment abolishes the antigenic capacity of gluten.
The reliable analysis of the cell cycle status has become increasingly relevant for scientific and clinical work, especially for the determination of tumor cell growth. One established method to ...characterize the proliferation activity of cells is the analysis of the Ki-67 protein. Ki-67 is expressed in the nucleus during the whole cell cycle except for the G0 phase. Several different protocols exist for the examination of the Ki-67 protein in tissue and cell culture, but most of them are defined for human cells. For the analysis of the Ki-67 protein in murine tissue and cell culture there is a variety of protocols existing which recommend different fixation and permeabilization reagents or special kits. In this study, we established a reliable protocol for Ki-67 staining in murine cells and tissue based on PFA fixation, which can be used not only for flow cytometry but also for immunofluorescence microscopy analysis. We tested our protocol successfully with three different Ki-67 anti-mouse antibodies in cell culture, regenerating liver tissue and mouse melanoma tumor to demonstrate the general applicability.
The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in ...tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.
An appropriate means of quantitating infectious Chlamydia from infected animals is essential for the evaluation of vaccines. However, unlike methods involving culture, nonculture methods, including ...detection of antigen or DNA, are not able to differentiate between viable and nonviable organisms. As an obligate intracellular bacterium, Chlamydia replicates inside host cells by forming unique organelles called inclusions. Here, we describe the enumeration of viable C. trachomatis from infected mice by culturing vaginal swabs on McCoy cells and counting inclusions via immunofluorescent microscopy.
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