Name: Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids (Video) | JoVE Journal
Uploaded: 2017-12-13T15:00:00.000+00:00
Duration: 9 min 51 s
Description: Learn immuno-fluorescent labeling of microtubules and centrosomal proteins in ex vivo intestinal tissue and 3D in vitro intestinal organoids. This method preserves delicate 3D architecture while maintaining antigenicity for accurate protein detection.

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5 minutes

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.).

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<li>van der Flier, L. G., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cells%2C+self-renewal%2C+and+differentiation+in+the+intestinal+epithelium%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Stem cells, self-renewal, and differentiation in the intestinal epithelium.</a> Annu Rev Physiol. 71, 241-260 (2009).</li>
<li>Goldspink, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ninein+is+essential+for+apico-basal+microtubule+formation+and+CLIP-170+facilitates+its+redeployment+to+non-centrosomal+microtubule+organizing+centres%5BTitle%5D)+AND+%22Open+Biol%22%5BJournal%5D)">Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres.</a> Open Biol. 7 (2), (2017).</li>
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<li>Andersson-Rolf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-step+generation+of+conditional+and+reversible+gene+knockouts%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">One-step generation of conditional and reversible gene knockouts.</a> Nat Methods. 14 (3), 287-289 (2017).</li>
<li>Schwank, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+repair+of+CFTR+by+CRISPR%2FCas9+in+intestinal+stem+cell+organoids+of+cystic+fibrosis+patients%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.</a> Cell Stem Cell. 13 (6), 653-658 (2013).</li>
<li>Goldspink, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+microtubule+end-binding+protein+EB2+is+a+central+regulator+of+microtubule+reorganisation+in+apico-basal+epithelial+differentiation%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">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/pubmed?term=((A+Simple+Device+to+Rapidly+Prepare+Whole+Mounts+of+the+Mouse+Intestine%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((A+video+protocol+of+retroviral+infection+in+primary+intestinal+organoid+culture%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((LIS1%2C+CLIP-170%27s+key+to+the+dynein%2Fdynactin+pathway%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Patterns+of+DNA+methylation+in+individual+colonic+crypts+reveal+aging+and+cancer-related+field+defects+in+the+morphologically+normal+mucosa%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">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/pubmed?term=((Establishment+of+conditionally+immortalized+epithelial+cell+lines+from+both+colon+and+small+intestine+of+adult+H-2Kb-tsA58+transgenic+mice%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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>
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<li>Rogers, S. L., Rogers, G. C., Sharp, D. J., Vale, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+EB1+is+important+for+proper+assembly%2C+dynamics%2C+and+positioning+of+the+mitotic+spindle%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle.</a> J Cell Biol. 158 (5), 873-884 (2002).</li>
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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

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.

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.

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

Research

JoVE Journal

Developmental Biology

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We describe a method to visualize GFP-labeled γδ 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.

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

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.

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

We present a step-by-step protocol for high-resolution, label-free, and three-dimensional imaging of organoids using low-coherence holotomography. This protocol details organoid culture preparation, imaging acquisition, and computational image analysis, enabling real-time visualization of structural dynamics and drug responses in living organoids.

Label-free, High-Resolution 3D Imaging and Machine Learning Analysis of Intestinal Organoids via Low-Coherence Holotomography

Learn immuno-fluorescent labeling of microtubules and centrosomal proteins in ex vivo intestinal tissue and 3D in vitro intestinal organoids. This method preserves delicate 3D architecture while maintaining antigenicity for accurate protein detection.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids (Video) | JoVE Journal

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

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