Name: using unfixed, frozen tissues to study natural mucin distribution
Uploaded: 2012-09-21T12:00:00
Duration: 11 min 39 s
Description: Watch this Scientific Journal Video about Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution at JoVE.com

The authors would like to thank Nicole M. Nemeth (University of Georgia) and Jeanne M. Fair (LANL) for their help in harvesting chicken tissues, and Steven A. Springer for his help during filming. The care of all birds in this study was in compliance with National Institutes of Health guidelines for the humane use of laboratory animals and all protocols were approved by the Institutional Animal Care and Use Committees at Los Alamos National Security, LLC, operator of the Los Alamos National Laboratory under Contract No. DE-AC52-06NA25396 with the U.S. Department of Energy. The care of mice in this study is in compliance with UCSD animal approved protocol. Human tissues were obtained as part of UCSD approved IRB protocol. This work was supported by grant 118645 from University of California Lab Fee President Program (P.G.) and grant NS047101 from National Institute of Neurological Disorders and Stroke (Neuroscience Microscopy Shared Facility, UC San Diego).

1. Tissue Embedding in OCT
<ol>
	<li>Prepare a freezing bath by adding dry ice to 2-methyl butane in a shallow Styrofoam box.</li>
	<li>Harvest the tissue, and gently damp on a tissue paper to dry excess liquid. If using snap-frozen tissue (tissue that was frozen in liquid nitrogen), allow the tissue to warm up to -20 &deg;C by placing it in a cryo-microtome chamber.</li>
	<li>Add a small amount of OCT to the Peel-A-Way freezing mold, just enough to cover the bottom of the mold.</li>
	<li>Place the tissue in the mold, make sure the tissue is rested on the bottom of the mold at the desired orientation. Once frozen, the tissue block will be sectioned either from the bottom or from the sides.</li>
	<li>Cover the tissue with OCT, and place the mold in the freezing bath. The OCT compound will turn white as the tissue freezes.</li>
	<li>Once frozen, peel the mold off the frozen block and place in a marked freezing bag.</li>
	<li>The frozen blocks can be kept at -80 &deg;C until use.</li>
</ol>
2. Tissue Sectioning
<ol>
	<li>Place tissue blocks in the cryo-microtome chamber, and allow them to reach -20 &deg;C (about 30 min).</li>
	<li>Cut a 3-5 &mu;m thick section, and place a positively charged glass slide on top of the section. The tissue will adhere to the slide.</li>
	<li>Air dry the tissues for 30-60 min.</li>
	<li>The slides can be used at this stage, or they can be kept at -80 &deg;C for future use.</li>
</ol>
3. Tissue Staining
<ol>
	<li>Slides that were stored at -80 &deg;C: allow the slides to thaw and air dry at room temperature for 30 min.</li>
	<li>Fix the slides with 10% buffered formalin for 30 min at room temperature.</li>
	<li>Wash three times in PBS or TBST buffer, for each wash dip the slides 10 times in 250 ml buffer. PBS buffer can be used for immunofluorescence staining, however, for chromogenic detection with Alkaline Phosphatase-conjugated antibody, TBST should be used since the phosphate in PBS inhibits Alkaline Phosphatase activity.</li>
	<li>Tissue sections slides are now ready to be stained.</li>
</ol>
4. Using Histochemical Stains such as Alcian Blue and Periodic Acid Schiff to Detect Mucus
<ol>
	<li>Alcian Blue stain:
	<ol>
		<li>Rinse the slides in water, incubate in 3% acetic acid for 3 min at room temperature.</li>
		<li>Stain with Alcian Blue pH 2.5 solution for 30 min at room temperature.</li>
		<li>Wash the slides in running tap water for 10 min, rinse in DI water.</li>
		<li>Counterstain in Nuclear Fast Red for 5 min at room temperature.</li>
		<li>Wash slides three times in DI water.</li>
	</ol>
	</li>
	<li>Periodic Acid Schiff stain:
	<ol>
		<li>Rinse the slides in water, incubate in freshly prepared 1% periodic acid for 5 min.</li>
		<li>Wash three times in DI water, dip once in miliQ water.</li>
		<li>Stain with Schiff reagent for 15 min at room temperature.</li>
		<li>Wash the slides in running tap water for 10 min, rinse in DI water.</li>
		<li>Counterstain in Surgipath Hematoxylin for 30 sec at room temperature.</li>
		<li>Wash slides three times in DI water.</li>
		<li>Incubate slides 30 sec in scott's tap water at room temperature.</li>
		<li>Wash three times in DI water.</li>
	</ol>
	</li>
	<li>Dehydrate and clear slides by incubating 1 min in 95% ethanol, followed by three quick changes in 100% ethanol, and three changes in Citrisolv, 2 min each. All at room temperature.</li>
	<li>Mount slides on coverslips with resinous medium (Cytoseal 60).</li>
</ol>
5. Using Lectins and Antibodies (Table 1) to Detect Glycan Epitopes by Histochemical Methods
<ol>
	<li>For fluorescence detection of three glycan epitopes using lectins, block the slides with 1% BSA in PBS for 10-30 min at room temperature.</li>
	<li>Since a biotinylated lectin is used, block endogenous biotin by incubating 15 min with 0.1% Avidin, followed by 15 min incubation with 0.01% biotin at room temperature.</li>
	<li>Wash the slides in PBS after each blocking step.</li>
	<li>Freshly prepare a mixture of 1 &mu;g/ml Rhodamine-conjugated succinylated wheat germ agglutinin (sWGA), 1.3 &mu;g/ml biotinylated Sambucus nigra agglutinin (SNA) and 5 &mu;g/ml Fluorescein-conjugated Jacalin in HEPES/NaCl buffer (10 mM HEPES, 150 mM NaCl pH 7.5).</li>
	<li>Place slides on a flat surface or in a staining box, and layer the lectin mixture on top. The mixture volume can be reduced by gently placing parafilm on the liquid, the parafilm flattens the liquid and prevents evaporation.</li>
	<li>Incubate 1 hr at room temperature in dark.</li>
	<li>Wash slides three times with PBS.</li>
	<li>Layer slides with 0.7 &mu;g/ml streptavidin-conjugated CY5 (to detect the biotinylated-SNA), and incubate for 30 min at room temperature in dark.</li>
	<li>Wash slides three times with PBS.</li>
	<li>Counterstain nuclei with 0.1 &mu;g/ml DAPI.</li>
	<li>Mount slides on coverslips with aqueous medium such as VectaMount mounting media (or any aqueous media).</li>
</ol>
6. Controlling for Lectin Staining Specificity
<ol>
	<li>Lectin staining specificity is controlled either by specific enzymatic cleavage of target glycan epitope prior to staining or by competition with small molecules.</li>
	<li>Enzymatic cleavage for Sambucus nigra agglutinin (SNA) binding specificity
	<ol>
		<li>Dilute Arthrobacter ureafaciens sialidase (AUS) to 250 mU/ml in 50 mM sodium acetate pH 5.5.</li>
		<li>Add water to the bottom of an empty tip-box, this will form a humid chamber during incubation.</li>
		<li>Place slides face up on the top tray of the tip-box, layer 150-200 &mu;l AUS solution on the slide and cover with a coverslip. Avoid air bubble formation.</li>
		<li>Close the box lid and incubate at 37 &deg;C for 2.5 hr.</li>
		<li>Wash slides three times in PBS at room temperature to remove all free sialic acids. These slides should be negative for SNA staining.</li>
	</ol>
	</li>
	<li>Competitive inhibitors for Jacalin and succinylated wheat germ agglutinin (sWGA) specificity
	<ol>
		<li>Aliquot 200 &mu;l of the lectin mixture prepared in step 5.4 to two Eppendorf vials.</li>
		<li>Add 200 mM Melibiose (Jacalin inhibitor) to one of the vials and Chitin-hydrolysate at 1:10 dilution (sWGA inhibitor) to the other vial.</li>
		<li>Overlay negative control slides with the inhibitors-containing mixture and incubate 1 hr at room temperature at the same time as the rest of the slides. These slides should be negative for Jacalin or sWGA staining, respectively.</li>
	</ol>
	</li>
</ol>
7. Representative Results
A comparison between tissue samples embedded in paraffin to frozen tissues embedded in cryo-protectant media (OCT) revealed striking difference in the preservation and quality of staining for mucin glycoproteins. Tissue staining with histochemical dyes, such as Alcian Blue and Periodic Acid Schiff, produce very different results in comparable tissue sections from frozen or paraffin embedded samples (Figure 1). It appears that the organic solvent (xylene or Citrisolv) that is used during the paraffin embedding process affects the distribution of secreted mucins on epithelia as well as removing much of the glycolipids from the samples (Figure 2). As a result, the mucus layer appears collapsed on the mucosa cells and is mostly found in goblet cells. Flash freezing of tissues in cryo-protectant media (OCT) maintained sample hydration and preserved the secreted mucins layer dimensions. The paraffin embedding process affected other mucus-associated glycans and glycolipids in a similar way. Glycan distribution was examined using lectins, which are routinely used for glycan detection (Figure 3) and antibodies against epitopes found on mucins and glycolipids (Figure 6). Because lectin binding is not well defined and is affected by the spatial distribution of glycans as well as the glycan structure14,15, it is important to apply the appropriate controls for lectin staining. Here we demonstrate two methods for controlling lectin staining on the tested tissues: enzymatic cleavage and competitive inhibition. Cleavage of the glycan epitopes was done by digesting the tissue section with glycan-specific enzymes, for example bacterial sialidase as control for sialic acid binding by SNA (Figure 4). In cases where specific enzyme (e.g. glycosidase) is not available for removing the glycan epitope studied, lectin specificity can be confirmed by adding a competitive inhibitor such as Melibiose for Jacalin staining or Chitin-hydrolysate for sWGA staining (Figure 5).
We demonstrate here that snap-frozen tissue samples, which are routinely obtained in the clinic and in research laboratories, can be further embedded in OCT and used to study mucin glycoproteins and the many glycans present on them.
<table border="1">
	<tbody>
		<tr>
			<td>Lectin / Ab</td>
			<td>Source</td>
			<td>Major specificity</td>
		</tr>
		<tr>
			<td>LFA</td>
			<td>Limax flavus (Yellow slug)</td>
			<td>Terminal Sia</td>
		</tr>
		<tr>
			<td>MAA*</td>
			<td>Maackia amurensis (Amur Maackia)</td>
			<td>Sia&alpha;2-3Gal&beta;1-R / 3-O-sulfate on Gal&beta;1-R</td>
		</tr>
		<tr>
			<td>SNA</td>
			<td>Sambucus nigra (Elderberry)</td>
			<td>Sia&alpha;2-6Gal / Sia&alpha;2-6GalNAc</td>
		</tr>
		<tr>
			<td>WGA</td>
			<td>Triticum vulgaris (Wheat germ)</td>
			<td>GlcNAc&beta;1-4GlcNAc&beta;1-4GlcNAc / Sia</td>
		</tr>
		<tr>
			<td>sWGA</td>
			<td>Succinylated Triticum vulgaris (Wheat germ)</td>
			<td>GlcNAc&beta;1-4GlcNAc&beta;1-4GlcNAc</td>
		</tr>
		<tr>
			<td>PNA</td>
			<td>Arachis hypogaea (Peanut)</td>
			<td>Gal&beta;1-3GalNAc (unmodified T-antigen)</td>
		</tr>
		<tr>
			<td>Jacalin</td>
			<td>Artocarpus integrifolia (Jacalin)</td>
			<td>Gal&beta;1-3GalNAc found on O-linked glycans</td>
		</tr>
		<tr>
			<td>ECA</td>
			<td>Erythrina cristagalli (Coral tree)</td>
			<td>Gal&beta;1-4GlcNAc</td>
		</tr>
		<tr>
			<td>TKH2</td>
			<td>Antibody</td>
			<td>Siaa2-6GalNAc(STn) on O-linked glycans</td>
		</tr>
		<tr>
			<td>CA19-9</td>
			<td>Antibody</td>
			<td>Siaa2-3Gal&beta;1-4(Fuca1-3)GlcNAc (SLea)</td>
		</tr>
		<tr>
			<td>SNH3</td>
			<td>Antibody</td>
			<td>Siaa2-3Gal&beta;1-3(Fuca1-4)GlcNAc (SLex)</td>
		</tr>
	</tbody>
</table>
Abbreviations: Ab, antibody; Sia, Sialic acid; Gal, Galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Fuc, Fucose; STn, Sialyl Tn; SLea, sialyl Lewisa; SLex, sialyl Lewisx. * Commercial sources for MAA include MALI, MALII &amp; MAH.
Table 1. A partial list of lectins and antibodies for glycan epitopes.
<img alt="Figure 1" src="/files/ftp_upload/3928/3928fig1.jpg" /> 
 Figure 1. Alcian Blue and Periodic Acid Schiff staining of frozen and paraffin-embedded colon tissues. Tissue sections of human, mouse or chicken colon specimens frozen in OCT (upper panel) or embedded in paraffin (lower panel) were stained with Periodic Acid Schiff (PAS) or Alcian Blue (AB). These reagents stain mucus pink or blue, respectively. Upper panel: In frozen tissues, in addition to mucins in goblet cells, secreted mucus was also visible (arrows). Lower panel: In paraffin-embedded tissues, mucus staining was confined to goblet cells. Nuclei were counterstained with Surgipath (PAS) or Mayer's (AB) Hematoxylin. Scale bar indicates 100 &mu;m. <a href="https://www.jove.com/files/ftp_upload/3928/3928fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3928/3928fig2.jpg" /> 
Figure 2. Citrisolv incubation results in substantial loss of mucins. Serial sections of frozen chicken ileum specimens were fixed in 10% buffered formalin and kept hydrated (left panel), dehydrated in ethanol by sequential incubation in 70%, 90% and 100% ethanol for 20 min each (middle panel), or dehydrated in ethanol and cleared with Citrisolv for 1 hr (right panel). Dehydrated samples were rehydrated back to PBS before Hematoxylin and Eosin (H&amp;E) or Alcian Blue staining. Ethanol dehydration and Citrisolv clearing improved tissue morphology (e.g., top row, middle and right images versus left image). Ethanol dehydration had no significant effect on Alcian Blue staining (middle row, compare left and middle images). In contrast, Citrisolv incubation reduced Alcian Blue staining and confined it to goblet cells (middle row, right image) in a pattern that was similar to that seen in paraffin-embedded tissues (Figure 1). These data imply that the strong staining of mucus granules in paraffin-embedded samples is due to shrinking and condensing of mucus in the goblet cells during the clearing step. The fainter and less dense staining of mucus in frozen OCT-embedded tissues reflects a more natural distribution of mucus in the tissue. Higher magnifications of boxed areas are marked with arrows. Scale bars indicate 50 &mu;m (top and middle rows) and 10 &mu;m (bottom row).
<img alt="Figure 3" src="/files/ftp_upload/3928/3928fig3.jpg" /> 
Figure 3. Binding of lectins to glycan epitopes on frozen and paraffin-embedded tissues. Chicken small intestine (ileum) specimens frozen in OCT (upper panels) or embedded in paraffin (lower panels) were probed with Jacalin (blue), sWGA (green) and SNA (red). Higher magnifications of boxed areas are marked with arrows. In frozen tissues Jacalin binding to O-linked glycans revealed structures that appeared to be oozing from goblet cells into the lumen (Jacalin, upper panel, arrows). In contrast, Jacalin binding to paraffin-embedded tissues was confined to goblet cells (Jacalin, lower panel, arrows) and to the villi brush border (bottom left image, arrow). sWGA staining of &beta;1-4GlcNAc partially co-localized with the binding of Jacalin lectin in both frozen tissues (upper panel, arrows), and in paraffin-embedded tissues (lower panel, arrows). In contrast, SNA lectin binding to &alpha;2-6 linked sialic acids is intracellular (SNA, arrowheads), and does not co-localize with Jacalin (color image, SNA in red marked with arrowhead, Jacalin in blue marked with dotted arrow). Scale bars indicate 100 &mu;m (left images) and 20 &mu;m (magnified boxed areas).
<img alt="Figure 4" src="/files/ftp_upload/3928/3928fig4.jpg" /> 
Figure 4. Enzymatic cleavage control for sialic acid staining with SNA. Chicken small intestine specimens were incubated with 250 mU/ml Arthrobacter ureafaciens sialidase (AUS) or with 50 mM sodium acetate pH 5.5 buffer for 2.5 h at 37 &deg;C. AUS treatment abolishes staining with biotinylated SNA, confirming SNA binding specificity to sialic acids. Scale bar indicates 100 &mu;m.
<img alt="Figure 5" src="/files/ftp_upload/3928/3928fig5.jpg" /> 
Figure 5. Competitive inhibition control for glycan staining with Jacalin and sWGA. Chicken small intestine (ileum) specimens were incubated with a Jacalin and sWGA mixture in the presence of specific lectin inhibitors: Melibiose (middle column), Chitin-hydrolysate (right column) or without inhibitor (A and D). Upper panel: (left) Jacalin staining without inhibitors. (center) Jacalin staining was inhibited by Melibiose. (right) Chitin-hydrolysate did not inhibit Jacalin staining. Lower panel: (left) sWGA staining without inhibitor. (center) Melibiose did not inhibit sWGA staining. (right) sWGA staining was inhibited by Chitin-hydrolysate. This inhibition confirms the specific interaction of lectins with the tissues. Asterisks mark image of staining inhibition. Scale bar indicates 100 &mu;m.
<img alt="Figure 6" src="/files/ftp_upload/3928/3928fig6.jpg" /> 
Figure 6. Detection of secretory mucins, glycolipids and glycan epitopes in frozen human colorectal cancer tissues. Colorectal cancer biopsies from villous carcinoma and mucous carcinoma were snap-frozen in liquid nitrogen and embedded in OCT. Tissue sections were incubated for 1 h with antibodies against the secreted mucin MUC5AC, sialyl Lewis a - glycan epitope found on gangliosides (colorectal cancer marker CA 19-9), and sialyl-Tn - abundant glycan epitope on mucins (detected with TKH2 antibody), followed by 30 min incubation with biotinylated donkey anti-mouse IgG secondary antibody, and 30 min incubation with peroxidase-conjugated streptavidin. Additional tissues were incubated for 1 hr with the biotinylated lectins SNA and sWGA, followed by 30 min incubation with peroxidase-conjugated streptavidin. Peroxidase staining was developed using AEC kit. Black scale bar indicates 200 &mu;m.

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No conflicts of interest declared.

Preservation of mucus and glycan epitopes in frozen tissues is superior to that of tissues that were embedded in paraffin. We demonstrated the preservation of secreted mucus layer (Figures 1 and 3) and the distribution of three glycans structures (Figure 3) in frozen tissues compared with paraffin-embedded tissues. Specialized fixatives, such as Carnoy's solution (60% ethanol, 30% chloroform, 10% acetic acid)17 have been developed for optimal preservation of the mucus layer in tissue samples. Optimally, this solution should be used to collect tissue samples that are dedicated for mucus studies and was shown to preserve the smooth appearance of mucus layer16-17. The mucus layer in unfixed frozen samples embedded in OCT appears rugged and in some areas may detach from the tissue, however the overall layer thickness is in agreement with that observed in tissues that were fixed with Carnoy's solution and embedded in paraffin16-17. For example, the mucus layer in frozen human colon tissue section is ~100 &mu;m (Figure 1), which is within the range reported for Carnoy's-fixed human colon sample 55.4&plusmn;2.5 &mu;m (range 7.7 - 204.8 &mu;m) 16.
It has been known for decades that ethanol dehydration results in ~30% shrinkage of biological samples18, and that organic solvents such as xylene, Citrisolv and chloroform extract lipids, glycolipids and, to some extent, proteins from the tissues13. Tissue processing for paraffin embedding includes the following steps: fixation (10% buffered formalin), dehydration (increasing ethanol concentration), and clearing (Citrisolv or xylene). By mimicking these steps on unfixed frozen tissue sections, we demonstrated that Citrisolv extracts mucus from frozen tissue sections resulting in tissue morphology that is similar to that of paraffin-embedded tissues (Figure 2, right panel). In contrast, the mucus layer was not altered by incubation with formalin or ethanol (Figure 2, left and center panels). This suggests that the clearing step of standard paraffin embedding procedure, which requires prolonged incubation in Citrisolv/xylene, results in the collapse of the mucus layer. Formalin fixation does not damage the mucus layer and frozen tissues sections that were fixed with formalin can readily be stained with lectins and antibodies against glycans, glycolipids and proteins (Figures 2, 3 and 6). These effects may be negligible for the study of membrane-bound proteins and tissue pathology, but they are devastating for highly hydrated structures such as the secreted mucus layer. However histological studies of mucins are still conducted mostly with paraffin-embedded samples, in which the mucus layer preservation is suboptimal. In depth analyses of mucus layer composition such as the precise identity of secreted or membrane bound MUC glycoprotein combinations require specific antibodies and mass spectrometry for identification of the protein backbones. Preservation of the mucus layer is but the initial requirement for such studies.
Many laboratories have tissue samples frozen in OCT that were collected in the past for different projects, these tissues can be readily used to study mucins, glycolipids and glycan distribution eliminating the need to collect tissues into special fixatives that are designed uniquely for mucus preservation. Frozen tissues undergo minimal processing and therefore the natural distribution of glycans, which are hydrated in nature, is preserved. This is especially important in the realm of microbial-host interactions. Knowledge of naturalistic distribution and abundance of secreted mucins and the many glycan structures decorating these "barrier" molecules will be key in understanding host defense, microbial exploitation and pathogenesis.

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent and equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>Fisher Scientific</td>
			<td>03551-4</td>
		</tr>
		<tr>
			<td>AEC peroxidase substrate kit</td>
			<td>Vector Labs</td>
			<td>SK-4200</td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Sigma-Aldrich</td>
			<td>A3157</td>
		</tr>
		<tr>
			<td>Anti-CA 19-9 monoclonal antibody</td>
			<td>Calbiochem</td>
			<td>CA1003</td>
		</tr>
		<tr>
			<td>Anti-MUC5AC monoclonal antibody</td>
			<td>Millipore</td>
			<td>MAB2011</td>
		</tr>
		<tr>
			<td>Avidin-Biotin blocking kit</td>
			<td>Vector Labs</td>
			<td>SP-2001</td>
		</tr>
		<tr>
			<td>Biotinylated donkey anti-mouse antibody</td>
			<td>Jackson Immunoresearch</td>
			<td>90863</td>
		</tr>
		<tr>
			<td>Biotinylated SNA</td>
			<td>Vector Labs</td>
			<td>B-1305</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
		</tr>
		<tr>
			<td>Chitin-hydrolysate</td>
			<td>Vector Labs</td>
			<td>SP-0090</td>
		</tr>
		<tr>
			<td>Cryostat microtome</td>
			<td>Leica Microsystems</td>
			<td>Leica CM 1800</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Surgipath Medical Ind.</td>
			<td>3801570</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Fisher Scientific</td>
			<td>H325-100</td>
		</tr>
		<tr>
			<td>Jacalin-FITC</td>
			<td>Vector Labs</td>
			<td>FL-1151</td>
		</tr>
		<tr>
			<td>Mayer's Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>MHS32</td>
		</tr>
		<tr>
			<td>Melibiose</td>
			<td>Sigma-Aldrich</td>
			<td>M5500</td>
		</tr>
		<tr>
			<td>Nuclear Fast Red</td>
			<td>Vector Labs</td>
			<td>H-3403</td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>VWR International</td>
			<td>25608-930</td>
		</tr>
		<tr>
			<td>Peroxidase conjugated streptavidin</td>
			<td>Jackson Immunoresearch</td>
			<td>94638</td>
		</tr>
		<tr>
			<td>Schiff reagent</td>
			<td>Electron microscopy sciences</td>
			<td>26052</td>
		</tr>
		<tr>
			<td>sWGA-Rhodamine</td>
			<td>Vector Labs</td>
			<td>RL1022S</td>
		</tr>
		<tr>
			<td>TKH2 monoclonal antibody</td>
			<td>ATCC</td>
			<td>HB-9654</td>
		</tr>
		<tr>
			<td>VectaMount aqueous mounting media</td>
			<td>Vector Labs</td>
			<td>H-5501</td>
		</tr>
		<tr>
			<td>Cytoseal 60</td>
			<td>Thermo Scientific</td>
			<td>8310-4</td>
		</tr>
		<tr>
			<td>Peel-A Way molds</td>
			<td>Polysciences Inc.</td>
			<td>18646A-1</td>
		</tr>
	</tbody>
</table>

using unfixed, frozen tissues to study natural mucin distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.
Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.
In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.
We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Watch this Scientific Journal Video about Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution at JoVE.com

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

using-unfixed-frozen-tissues-to-study-natural-mucin-distribution

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address existing knowledge gaps. This protocol represents a demonstrated method for nondestructively sampling rare butterflies for population genetic structure or DNA barcoding analyses. It uses the chorion of hatched butterfly ovae to yield sufficiently high quantity and quality DNA for successful gene sequencing to confirm species identity and quantify genetic variation. It may be particularly useful when other tissue sampling techniques are impractical or unavailable. While developed for a lepidopteran, it nonetheless could easily be adapted for use with other insect species. It was specifically designed with ease of use as a goal to help maximize broad implementation by individuals of varying experience and skill levels, such as community scientists, conservation practitioners, and students, and for use over large geographic areas to facilitate broad population sampling. The data generated can help inform taxonomic and listing decisions, conservation and management actions, and enhance basic ecological research.

Here, we present a straightforward protocol for noninvasive genetic sampling of butterfly populations based on the field collection of residual egg debris. It can be used to confirm species identity and quantify genetic variation. This protocol can be easily adapted to broader groups for community science involvement.

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address ...

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.