Name: immuno-fluorescent labeling of microtubules and centrosomal proteins in ex vivo intestinal tissue and 3d in vitro intestinal organoids
Uploaded: 2017-12-13T15:00:00
Duration: 9 min 51 s
Description: 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

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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G., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+self-renewal,+and+differentiation+in+the+intestinal+epithelium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ninein+is+essential+for+apico-basal+microtubule+formation+and+CLIP-170+facilitates+its+redeployment+to+non-centrosomal+microtubule+organizing+centres.">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><li>Verissimo, C. 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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&#8217;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 &#956;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&#8211;38% in H2O)</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 
			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.

Isolation of intestinal tissue for immuno-labeling 
The described tissue isolation protocols for colon and small intestine were optimized for preservation and immuno-labeling of microtubules and associated proteins, but not for stem cell viability and organoid generation (Figure 1 and Table 1). The aim was to generate crypt and villus factions that were as clean (devoid of mucus and other tissue) as possible, while minimizing exposure to EDTA and cold to preserve structure and prevent depolymerization of microtubules with ice cold solutions, which induce depolymerization of all but stable microtubules. Figure 2 shows examples of images of Fractions 2 and 3 from isolated small intestinal tissue, with Fraction 2 containing a mixture of both villi and crypts (Figure 2A, B), while Fraction 3 contains mainly crypts (Figure 2C, D).
Fixation and immuno-labeling of isolated intestinal tissue 
The individual or combined fractions were then processed for fixation and immuno-labeled through a series of steps including fixation, detergent, blocking, antibody, and washing solutions, before re-suspending the final villi/crypts pellet in mounting media, transferring to slides, and covering with glass coverslips. The crypts and villi were then imaged on a confocal microscope.
Good preservation and labeling of microtubules and actin in both villi and crypts was achieved by the following: a combination of formaldehyde/methanol fixation at -20 &#176;C, repeated washing in PBS containing 0.1% detergent and 1% serum and blocking in PBS with 0.1% detergent and 10% serum, followed by overnight incubation at 4 &#176;C in primary antibodies and then 2 h in secondary antibodies at room temperature (Figure 3). Formaldehyde/methanol fixation also worked well for labeling +TIPs such as the EBs and CLIP-170 in isolated crypts and villi (Figure 4). EB3 accumulations at the plus-end of microtubules (known as comets) were evident in crypts (Figure 4A), while association along the lattice of stable microtubules could be seen in villi samples (Figure 4C). Distinct localization of CLIP-170 and p150Glued (subunit of dynactin) was clearly evident at the apical n-MTOCs in isolated villi (Figure 4B). Fixation with the formaldehyde/methanol protocol did not consistently work for ninein localization in isolated intestinal tissue using our Pep3 antibody against mouse ninein. However, methanol fixation at -20 &#176;C followed by the same washing and blocking solutions as for formaldehyde/methanol gave very good localization of ninein within isolated crypts and villi (Figure 5; reference8). Interestingly, while ninein is concentrated at the apical centrosomes some accumulation at the cell base was evident in some cells within isolated crypts (Figure 5). Whether this is due to non-specific labeling or a consequence of the isolation procedure delaying fixation (and thus affecting preservation) will need further investigation. However, methanol-fixed (-20 &#176;C) cryostat sections of villi (see Figure 3bi in8) also revealed ninein at the cell base in some cells suggesting that ninein may also associate with a basal population of microtubules.
Fixation and immuno-labeling of organoids isolated from basement matrix 
Small intestinal organoids were generated and grown in basement matrix for three weeks or longer (Figure 6A; reference6,15). A cold (4 &#176;C) cell recovery solution was used to isolate organoids from the basement matrix. The depolymerized basement matrix solution with organoids was transferred to tubes and centrifuged prior to fixation and immuno-labeling. This produced very clean preparations and allowed good access to the organoids for the various solutions. The differentiated cells within the organoid villus domains contain stable apico-basal microtubules; these labeled well in most cases (Figure 6B, C) and EB1 could also be seen along the microtubule lattice (Figure 6E; reference13). However, the cold cell recovery solution may result in depolymerization of the dynamic microtubules, which was evident in some samples by the lack of EB1 comets (which bind to the plus-end of growing microtubules) within the basal crypt domains (Figure 6F). In other samples, astral (dynamic) microtubules were preserved (Figure 6D). Organoid isolation prior to fixation and immuno-labeling also worked for junctional proteins, ninein, CLIP-170, and cell markers, such as mucin for goblet cells and chromogranin A for enteroendocrine cells.
Fixation and immuno-labeling of organoids within basement matrix 
Figure 7 shows an organoid at the cyst stage (A-C) and at an early stage of crypt development (D), both fixed in formaldehyde/methanol and immuno-labeled for microtubules and ninein. Good microtubule preservation and labeling as well as labeling for ninein at apical n-MTOCs were evident. Figure 8A, B shows a crypt domain within a day 6 organoid fixed with the methanol protocol and labeled for microtubules and EB1. Good preservation of microtubules and EB1 comets was evident suggesting preservation of dynamic microtubules.
Organoids were also fixed and immuno-labeled while remaining within the basement matrix. The disadvantages of this procedure may be poor penetration of fixative and trapping of antibodies within the basement matrix (Figure 8B), although in both cases less frequently when 0.1% detergent was included in the fixative and/or wash solutions. In addition, 4% PFA did not preserve the basement matrix well but caused it to dissolve, although this was less so with 1% PFA. Methanol fixation, on the other hand, sometimes induced organoid collapse.
Labeling with some antibodies such as against the stem cell marker Lgr5 and Paneth cell marker CD24 proved unsuccessful with the 4% PFA, methanol, or formaldehyde/methanol protocols. However, fixing the organoids within the basement matrix with 1% PFA in PBS with 0.1% detergent at room temperature did result in labeling for both Lgr5 and CD24 (Figure 9).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56662/56662fig1.jpg" /> 
Figure 1: Isolation of small intestinal villi and crypts. Flow diagram of the key steps in small intestinal villus and crypt isolation prior to fixation and immuno-labeling. <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56662/56662fig2.jpg" /> 
Figure 2: Isolated villi and crypts from mouse small intestine. Brightfield microscope images of intestinal fractions showing villi (large arrows) and crypts (small arrows). (A, B) Fraction 2 contains a mixture of villi and crypts, and preservation of the morphology on the villus and crypt is evident in B. (C, D) Fraction 3 shows isolation of crypts and absence of villi and intact crypts including a bifurcated crypt in C. Scale bars = 500 &#956;m (A, C); 100 &#956;m (B, D). <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56662/56662fig3.jpg" /> 
Figure 3: Isolated small intestinal villus and crypt fixed in formaldehyde/methanol and immuno-labeled for microtubules and actin. (A) Schematic of the villus and crypt epithelium with different cell types indicated. The highlighted boxes indicate the representative regions imaged in B and C. (B, C) Confocal optical sections through part of a villus (B) and basal crypt (C) isolated from the small intestine using 30 mM EDTA and fixed in formaldehyde/methanol, washed in PBS containing 1% goat serum and 0.1% detergent, blocked in PBS containing 10% goat serum and 0.1% detergent, and labeled for microtubules with rat monoclonal anti-tubulin antibody (green) and for actin with rabbit polyclonal anti-&#946;-actin antibody (red). Well preserved apico-basal microtubule bundles are evident in both villus and crypt cells, and actin can be seen concentrated in the apical region facing the lumen (arrow). Scale bars = 5 &#956;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56662/56662fig4.jpg" /> 
Figure 4: Isolated small intestinal crypt and villi fixed in formaldehyde/methanol and immuno-labeled for microtubules, EB3, p150Glued, and CLIP-170. Confocal optical sections of crypt and villi regions isolated from the small intestine using 30 mM EDTA and fixed in formaldehyde/methanol, washed in PBS containing 10% goat serum and 0.1% detergent, blocked in PBS containing 10% goat serum and 0.1% detergent, and immuno-labeled. (A) Crypt labeled with rabbit polyclonal &#945;-tubulin antibody (red) and rat monoclonal EB3KT36 antibody (green) and stained for DNA with DAPI (blue) showing apico-basal microtubules and EB3 comets. The inverted single channel image clearly shows EB3 comets throughout the basal crypt cells suggesting good preservation of dynamic as well as stable microtubules. (B) Villus epithelial cells labeled with rabbit polyclonal CLIP-170 antibody (red, see also reference16) and mouse monoclonal p150Glued antibody (green) showing apical co-localization. Inverted single channel images are shown below. (C) Villus cells labeled with rabbit polyclonal &#945;-tubulin antibody (red) and rat monoclonal EB3-KT36 antibody (green) and stained for DNA with DAPI (blue) showing apico-basal microtubules with EB3 along the lattice. The EB3 lattice association is highlighted in the enlarged image while the inverted single channel image suggests both EB3 comets and lattice association. Scale bars = 5 &#956;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56662/56662fig5.jpg" /> 
Figure 5: Isolated colon crypt fixed in methanol, immuno-labeled for ninein and E-cadherin, and stained with DAPI. Confocal optical section of the basal and transit-amplifying region of a crypt isolated from the colon using 3 mM EDTA and fixed in methanol, washed in PBS containing 1% goat serum and 0.1% detergent, and blocked in PBS containing 10% goat serum and 0.1% detergent. The crypt was labeled with rabbit polyclonal ninein antibodies (Pep3, see also reference8, red) and mouse monoclonal E-cadherin antibody (green) and stained with DAPI (blue). The inverted single channel image shows ninein only. The image shows a well-preserved crypt with E-cadherin revealing the outline of the individual cells and ninein concentrated at the apical centrosomes. It suggests good penetration of fixative and antibodies, and preservation of antigenicity. Scale bar = 5 &#956;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56662/56662fig6.jpg" /> 
Figure 6: Organoid development, fixation, and immuno-labeling of isolated organoids. (A) Phase contrast images showing different stages of organoid development from cell aggregates to cyst with bud initiation and fully formed organoids with crypt and villus domains. (B-F) Confocal optical sections through organoids isolated from basement matrix using cell recovery solution at 4 &#176;C (10 min) followed by formaldehyde/methanol fixation, washing in PBS containing 10% goat serum and 0.1% detergent, blocking in PBS containing 10% goat serum and 0.1% detergent, and immuno-labeling for microtubules, &#946;-catenin, and EB1. (B) Organoid cyst labeled for microtubules (blue) and &#946;-catenin (red) revealing good microtubule preservation and labeling in most cells. (C) Distinct apico-basal microtubules are evident in these enlarged epithelial cells from an organoid cyst. (D) Dividing cells labeled for microtubules showing spindles including astral (dynamic) microtubules (arrow). (E, F) Villus domain (E) and crypt domain (F) organoid regions showing some EB1 labeling along the lattice of stable microtubules, especially in the villus, while very few EB1 comets are seen even within the basal crypt suggesting that dynamic microtubules have not been preserved. Scale bars = 20 &#956;m (A); 2 &#956;m (D); 5 &#956;m (B, C, E-F). <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56662/56662fig7.jpg" /> 
Figure 7: Organoids fixed within the basement matrix in formaldehyde/methanol and immuno-labeled for microtubules and ninein. Confocal optical sections of organoids fixed in formaldehyde/methanol, washed and blocked in PBS containing 10% goat serum and 0.1% detergent, and labeled while remaining in the basement matrix. (A-C) Organoid cyst labeled for microtubule (green) and ninein (Pep3; reference8, red) and stained with DAPI (blue) showing the merged image in A and inverted single channel images for microtubules (B) and ninein (C). The images show apico-basal microtubules and apical ninein localization, suggesting very good structural preservation of the organoid and penetration of antibodies as well as clearing of unbound antibodies. (D, E) Organoid with developing crypt fixed and labeled as above and again showing excellent structural preservation, labeling, and clearing of antibodies. Distinct apico-basal microtubules and apical n-MTOC ninein localization is evident and highlighted in the enlarged image (E) of the boxed region in D. Scale bars = 10 &#956;m (A-D); 5 &#956;m (E). <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56662/56662fig8.jpg" /> 
Figure 8: Organoids fixed within the basement matrix in methanol and immuno-labeled for microtubules and EB1. Confocal optical sections of organoids fixed in methanol, washed and blocked in PBS containing 10% goat serum and 0.1% detergent, and labeled, while remaining in the basement matrix. (A) Cyst domain from a fully developed organoid labeled with rabbit polyclonal &#945;-tubulin (red) and mouse monoclonal EB1 (green) antibodies showing apico-basal microtubules, spindles (arrow) in two dividing cells and distinct EB1 comets. Some trapping of unbound EB1 antibodies is evident. However, good structural preservation and labeling of microtubules and EB1 is observed. The presence of EB1 comets suggests that dynamic microtubules have been preserved (A, invert). (B) Inverted image of organoid cyst region showing &#945;-tubulin antibody labeling with considerable antibodies trapped within the surrounding basement matrix (arrow). Scale bars = 5 &#956;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56662/56662fig9.jpg" /> 
Figure 9: Organoids fixed within the basement matrix in 1% PFA and immuno-labeled for Lgr5 and CD24. Confocal optical sections of organoids fixed within the basement matrix in 1% PFA in PBS containing 0.1% detergent, washed in PBS with 1% goat serum and 0.1% detergent, and labeled with antibodies against Lgr5 and CD24. (A, B) Stem cell niche within a crypt domain showing Paneth cells positive for CD24 (red). The confocal and phase contrast images have been merged in A. B shows the single channel of CD24 labeling. (C) Stem cell region within a crypt domain showing a Lrg5 positive stem cell. Scale bars = 10 &#956;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/56662/56662table1.jpg" /> 
Table 1: Timeline of small intestinal crypt and villi isolation and fixation. <a href="https://cloudflare.jove.com/files/ftp_upload/56662/56662table1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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