The authors thank Paul Thomas for microscope advice and assistance. This work involved here was supported by the BBSRC (grant no. BB/J009040/1 to M.M.M. and T.W.).

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microtubule+end-binding+protein+EB2+is+a+central+regulator+of+microtubule+reorganisation+in+apico-basal+epithelial+differentiation.">The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation.</a> Journal of Cell Science. 126 (17), 4000-4014 (2013).</li><li>Yoneda, M., Molinolo, A. A., Ward, J. M., Kimura, S., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Device+to+Rapidly+Prepare+Whole+Mounts+of+the+Mouse+Intestine.">A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine.</a> J Vis Exp. (105), e53042 (2015).</li><li>Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+video+protocol+of+retroviral+infection+in+primary+intestinal+organoid+culture.">A video protocol of retroviral infection in primary intestinal organoid culture.</a> J Vis Exp. (90), e51765 (2014).</li><li>Coquelle, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIS1,+CLIP-170's+key+to+the+dynein/dynactin+pathway.">LIS1, CLIP-170's key to the dynein/dynactin pathway.</a> Mol Cell Biol. 22 (9), 3089-3102 (2002).</li><li>Belshaw, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+DNA+methylation+in+individual+colonic+crypts+reveal+aging+and+cancer-related+field+defects+in+the+morphologically+normal+mucosa.">Patterns of DNA methylation in individual colonic crypts reveal aging and cancer-related field defects in the morphologically normal mucosa.</a> Carcinogenesis. 31 (6), 1158-1163 (2010).</li><li>Whitehead, R. H., VanEeden, P. E., Noble, M. D., Ataliotis, P., Jat, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+conditionally+immortalized+epithelial+cell+lines+from+both+colon+and+small+intestine+of+adult+H-2Kb-tsA58+transgenic+mice.">Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.</a> Proc Natl Acad Sci U S A. 90 (2), 587-591 (1993).</li><li>Hamilton, K. L., Butt, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+transport+into+everted+sacs+of+the+small+intestine+of+mice.">Glucose transport into everted sacs of the small intestine of mice.</a> Adv Physiol Educ. 37 (4), 415-426 (2013).</li><li>Rogers, S. L., Rogers, G. C., Sharp, D. J., Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+EB1+is+important+for+proper+assembly,+dynamics,+and+positioning+of+the+mitotic+spindle.">Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle.</a> J Cell Biol. 158 (5), 873-884 (2002).</li></ol>

The authors declare no competing financial interests.

Isolation of intestinal tissue 
Isolation of small intestinal crypts and villi and colonic crypts involves exposing the mucosal surface, treatment with EDTA solution to loosen cell contacts, fractionation (shaking), and centrifugation. The presented intestinal villi/crypt isolation protocol has been modified from Belshaw et al. and Whitehead et al.17,18
Exposing the mucosal surface 
We have experimented with a number of approaches to expose the mucosal surface of the intestinal tract in the development of this procedure. A classic approach is to evert (turn inside out) the tube, usually in segments about 100 mm long, using a metal rod that is caught in a fold of the tissue at one end and then the remaining tube slid over the tube19. For mouse tissue, a metal rod (2.4 mm diameter) with rounded ends is ideal. This approach has the benefit of expanding the mucosal surface allowing better access to PBS and EDTA. We initially used this approach but moved to cutting the tube into short lengths (about 5 cm) and opening each section with dissecting scissors as this proved easier. This approach is appropriate if only a few intestines are required; but if more animals were to be used in an experiment then a purpose-built device for cutting open the tube longitudinally, as described by Yoneda et al.14 would be more efficient.
Detachment of villi and crypts from the muscle layer 
Initially we used 3 mM EDTA in PBS and relatively long incubation times of up to 60 min to loosen the mucosal surface from the underlying tissue17,18. At this concentration of EDTA we found an incubation time of 30 min was sufficient to loosen crypts from mouse colon. However, for the small intestine crypt/villus isolation we tried using more concentrated EDTA for a shorter time, which proved to be an efficient approach. All subsequent work was undertaken with tissue extracted using the 30 mM EDTA technique generating relevant fractions for villi or crypts. For crypts, we normally pooled fractions 3 - 5 before fixing but it is important to check whether these are the appropriate fractions as the timings will depend on a number of factors such as position along the intestinal tract, age of mouse, inflammation, previous diet, etc. Similarly, the length of time the tissue needs to be shaken following the EDTA treatment to be effective may vary under different conditions. The result is isolated tissue fractions containing a mixture of villi and crypts or mainly either villi or crypts (Figure 2). As there are no villi in the colon, the crypt extraction may be achievable in one step by shaking the tissue in the tube for 30 s. These fractions can then be fixed and processed for immuno-labeling.
Isolation of intestinal organoids from basement matrix 
Isolation of organoids from basement matrix domes can be achieved by using cell recovery solution. The solution works by depolymerizing the gelled basement matrix but the temperature needs to be 2 - 8 &#176;C. A note of caution is that dynamic microtubules may not be preserved. Thus, for immuno-labeling of dynamic microtubules and +TIPs such as the EBs cell, recovery from basement matrix prior to fixation is not recommended. However, most of the microtubules in the differentiating organoid cells are relatively stable and these were preserved (Figure 6). It also worked well for immuno-labeling of centrosomal and junctional proteins as well as cell markers.
Fixation protocols 
Formaldehyde (freshly made from PFA) is a relatively rapid-acting fixative that forms reversible cross-links and 4% PFA works well for example, in immuno-labeling microtubules and gamma-tubulin and staining actin filaments with Phalloidin. More dilute PFA solutions such as 1% worked well for immuno-labeling for example, with the stem cell markers Lgr5 and Paneth cell marker CD24 within the crypt stem cell niche, while higher concentrations of PFA did not work.
The addition of glutaraldehyde gives better preservation of microtubules and the so called PHEMO fixation which consists of a mixture of 3.7% PFA, 0.05% glutaraldehyde and 0.5% detergent in PHEMO buffer (68 mM PIPES, 25 mM HEPES, 15 mM EGTA and 3 mM MgCl2)2 gives excellent preservation of microtubules without compromising antigenicity. It also works well for immuno-labeling gamma-tubulin, &#946;-catenin, and E-cadherin, and staining actin filaments with phalloidin. However, in 3D tissue and organoid cultures, the PHEMO fixation produced inconsistent results and was therefore not used.
Methanol is a coagulant fixative that gives relatively good penetration and tends to preserve antigenicity. Fixation with 100% methanol (-20&#160;&#176;C) introduces some shrinkage, gives moderate morphology preservation, and works for microtubules, +TIPs, and many centrosomal antibodies including ninein in 2D cell cultures. However, some organoids collapsed when using this fixation method. In addition, penetration of antibodies through entire crypts, villi, or organoids was initially a problem but the addition of 0.1% detergent to the wash solution and prolonged washing achieved better results.
A combination of formaldehyde and methanol had previously been used by Rogers et al.20 to immuno-label EB1 in Drosophila. A fixation protocol based on a mixture of formaldehyde and methanol was therefore developed for intestinal tissue and organoids based on 3% formaldehyde and 97% methanol chilled to -20 &#176;C, but omitting the 5 mM sodium carbonate from the mixture that was used by Rogers et al.20 In addition, samples were fixed in the freezer at -20 &#176;C. This worked particularly well for immuno-labeling +TIPs, such as CLIP-170 and the EBs, but also proved excellent for fixing and immuno-labeling microtubules and actin within tissue and 3D organoids. Very good structural preservation was evident and antigenicity was preserved for several cytoskeletal and associated proteins as well as centrosomal proteins such as gamma-tubulin and ninein, although labeling for ninein worked more consistently with methanol fixation.

Formation of epithelia with apico-basal polarity is a fundamental process in development and involves a dramatic reorganization of the microtubules and centrosomal proteins. A radial microtubule array emanating from a centrally located centrosomal microtubule organizing center (MTOC) is prominent in many animal cells and this is well suited for relatively flat cells. In contrast, columnar epithelial cells, such as those of the intestine, assemble non-radial transcellular microtubule arrays that better support the shape and specialized functions of these cells. This dramatic reorganization of the microtubules is achieved by the centrosome moving to the apex and apical non-centrosomal MTOCs (n-MTOCs) forming, which becomes responsible for anchorage of the transcellular microtubules1,2,3,4,5.
Much of our knowledge of epithelial differentiation and the associated microtubule reorganization has come from investigations of 2D in vitro cell layers that do not display the in vivo tissue architecture. Development of 3D in vitro organoid cultures, pioneered by Clevers and co-workers6, represents a major technological advancement as they mimic in vivo architecture and development. A hierarchy of epithelial differentiation is evident in the intestine; stem cells at the bottom of crypts give rise to immature transit amplifying cells that proliferate and gradually differentiate as they migrate up the crypt onto the small intestinal villus or colonic surface, where they become fully differentiated prior to being shed into the lumen7. Importantly, this is replicated in intestinal organoids where cells from the stem cell niche proliferate forming cysts that subsequently generate crypt-like buds with stem cells at the bottom and differentiation gradually progressing towards the cyst region, which becomes villus-like8. The intestinal organoid therefore represents a powerful model to study not only microtubule and centrosomal reorganization during epithelial differentiation but numerous other proteins, as well as providing an ideal platform for screening of drugs and food compounds of potential therapeutic benefits9,10.
Organoids are well suited for live-imaging of fluorescent-tagged proteins and both knock-in and knock-out organoids can be generated using CRISPR/Cas9 gene editing11,12. However, establishing the expression and localization of the endogenous proteins to be studied is important, especially to verify the behavior of the tagged proteins. Immuno-labeling 3D organoids grown in basement matrix or ex vivo isolated tissue is more complex than cells grown in culture dishes in 2D. The fixation protocol needs to preserve the delicate 3D architecture of organoids while still preserving antibody antigenicity (i.e., the epitopes for binding antibodies). For example, 4% paraformaldehyde (PFA) is commonly used as a fixative but while it is a relatively rapid acting fixative and gives good morphological preservation, in our experience it frequently results in loss of antigenicity and is not suitable for many centrosomal antibodies. The ability of the fixative and antibodies to penetrate 3D structures and tissue should also be considered. To this end, we have modified and developed protocols for tissue isolation and indirect immuno-labeling of 3D in vitro organoids and ex vivo isolated intestinal tissue. We describe how to isolate small intestinal crypts and villi and colonic tissue, and include a protocol for isolation of 3D organoids as an alternative to fixing and immuno-labeling within the basement matrix. We present three alternative fixation protocols for immuno-labeling of microtubules and centrosomal proteins, such as ninein, and microtubule plus-end tracking proteins (+TIPs), such as the EB proteins and CLIP-170 (see also references8,13). We also discuss the pros and cons associated with each protocol.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>D8537-500ML</td><td>washing and PFA</td></tr><tr><td>Cell Recovery Solution</td><td>Corning</td><td>354253</td><td>isolate organoids</td></tr><tr><td>lobind microcentrifuge tubes</td><td>Eppendorf</td><td>30108116</td><td>prevent cells sticking</td></tr><tr><td>0.5 M EDTA solution, pH 8.0</td><td>Sigma</td><td>03690-100ML</td><td>Crypt isolation</td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Fisher Scientific</td><td>11517532</td><td>Isolate crypts from villi</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td>detergent</td></tr><tr><td>goat serum</td><td>Sigma</td><td>G6767</td><td>blocking agent</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td></td><td>used as non-stick agent</td></tr><tr><td>Paraformaldehyde, 4% in PBS</td><td>Alfa Aesar</td><td>J61899</td><td>fixative</td></tr><tr><td>Formaldehyde solution (36.5&amp;ndash;38% in H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma</td><td>F8775</td><td>used as fixative</td></tr><tr><td>Methanol 99.9% Analytical grade</td><td>Fisher</td><td>M/4000/17</td><td>used as fixative</td></tr><tr><td>MaxFluor Mouse on Mouse Immunofluorescence Detection Kit</td><td>Maxvision&lt;br/&gt; Biosciences</td><td>MF01-S</td><td>commercial immunofluorescence kit; to reduce non-specific labelling</td></tr><tr><td>Rotator SB2 or SB3</td><td>Stuart</td><td></td><td>microcentrifuge tube rotator</td></tr><tr><td>Sodium citrate</td><td></td><td></td><td>antigen retrieval</td></tr><tr><td>Hydromount</td><td>National Diagnostics</td><td>HS-106</td><td>mountant</td></tr><tr><td>DABCO - 1,4-diazabicyclo[2.2.2]octane</td><td>Sigma</td><td>D27802</td><td>anti-fade agent</td></tr><tr><td>Permanent Positive Charged, Pre-Washed, 90 Degree Ground Edges</td><td>Clarity</td><td>N/C366</td><td>slides</td></tr><tr><td>Glass coverslip - thickness no. 1, 22 x 50 mm</td><td>Clarity</td><td>NQS13/2250</td><td>coverslips</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>Basement matrix for 3D organoid culture</td></tr><tr><td>50 mL conical tubes</td><td>Sarstedt</td><td>62.547.254</td><td>for processing tissue/organoids</td></tr><tr><td>15 mL conical tubes</td><td>Sarstedt</td><td>62.554.502</td><td>for processing tissue/organoids</td></tr><tr><td>1.5 mL LoBind tubes</td><td>Eppendorf</td><td>30108051</td><td>for isolated organoids</td></tr><tr><td>Mouse monoclonal anti-E-cadherin antibody</td><td>BD Biosciences</td><td>610181</td><td>primary antibody 1:500</td></tr><tr><td>Rat monoclonal anti-tubulin YL1/2 antibody</td><td>Abcam</td><td>ab6160</td><td>primary antibody 1:100</td></tr><tr><td>Rabbit alpha-tubulin antibody</td><td>Abcam</td><td>ab15246</td><td>primary antibody 1:100</td></tr><tr><td>Rabbit anti-beta-actin antibody</td><td>Abcam</td><td>ab8227</td><td>primary antibody 1:100</td></tr><tr><td>Rat monoclonal anti-p150Glued antibody</td><td>BD Bioscience</td><td>610473/4</td><td>primary antibody 1:100</td></tr><tr><td>Rat monoclonal EB3KT36 antibody</td><td>Abcam</td><td>ab53360</td><td>primary antibody 1:200</td></tr><tr><td>Mouse monoclonal EB1 antibody</td><td>BD Bioscience</td><td>610535</td><td>primary antibody 1:200</td></tr><tr><td>Rabbit anti-Lgr5 antibody</td><td>Abgent</td><td>AP2745d</td><td>primary antibody 1:100</td></tr><tr><td>Dylight anti-rat 488</td><td>Jackson</td><td>112545167</td><td>seconday antibody 1:400</td></tr><tr><td>Dylight anti-mouse 488</td><td>Jackson</td><td>ST115545166</td><td>seconday antibody 1:400</td></tr><tr><td>Dylight anti-rabbit 647</td><td>Jackson</td><td>111605144</td><td>seconday antibody 1:400</td></tr></tbody></table>

immuno-fluorescent labeling of microtubules and centrosomal proteins in ex vivo intestinal tissue and 3d in vitro intestinal organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Watch this Scientific Journal Video about Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids at JoVE.com

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

immuno-fluorescent-labeling-microtubules-centrosomal-proteins-ex-vivo

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JoVE Journal

Developmental Biology

15.3K Views.  University of East Anglia. Presenteren wij protocollen voor isolatie van intestinale 3D structuren uit weefsel in vivo en in vitro kelder matrix ingesloten organoids, en detail verschillende fixatie en kleuring van protocollen die zijn geoptimaliseerd voor immuno-labeling van microtubuli, centrosomal en regulates eiwitten zo goed zoals markeringen met inbegrip van de stamcel proteïne Lgr5 cel.

Video: Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Medicine

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Immunology and Infection

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Biology

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

Efficient Swiss Roll Method for High-Quality Immunostaining of Intestinal Tissue

Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.

Author Spotlight: Effective Immunofluorescent Staining Protocol for Intestinal Cell Biology Research

The intestine is vital for digestion and absorption. Each region-duodenum, jejunum, ileum, colon-serves distinct functions due to unique cellular structures. Studying intestinal physiology demands meticulous tissue analysis. This protocol outlines tissue fixation and processing using the Swiss roll technique, ensuring accurate immunostaining through proper tissue preservation and orientation.

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ...

An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections

Source: Mizutani, Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57475">Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections.</a>&#160;J. Vis. Exp. (2018)
This video illustrates an immunofluorescence-based method for visualizing intestinal tuft cells in free-floating mouse jejunum cryosections. Within the intestinal villi, the co-localization of green fluorescence at the luminal tip with an extended red rootlet mass indicates the presence of tuft cells.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

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