Name: using robotic systems to process and embed colonic murine samples for histological analyses
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses at JoVE.com

We thank the department of Pathology of the IRCCS Policlinico Hospital, Milan for technical support and the IEO Animal Facility for assistance in animal husbandry.

Animal procedures were approved by the Italian Ministry of Health (Auth. 127/15, 27/13) and followed the animal care guidelines of the European Institute of Oncology IACUC (Institutional Animal Care and Use Committee)
1. Chronic Colitis Induction by Repetitive DSS Administration
<ol>
	<li>Separate age and sex matched mice in 2 groups (treatment DSS vs. control H2O, at least 5 mice littermates per experimental group).</li>
	<li>Administer 2.5% DSS (40 kDa) in the drinking water for...

<ol>
	<li>Gibson-Corley, K. N., 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=Successful+Integration+of+the+Histology+Core+Laboratory+in+Translational+Research.">Successful Integration of the Histology Core Laboratory in Translational Research.</a> Journal of histotechnology. 35, 17-21 (2012).</li><li>Olivier, A. K., 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=Genetically+modified+species+in+research:+Opportunities+and+challenges+for+the+histology+core+laboratory.">Genetically modified species in research: Opportunities and challenges for the histology core laboratory.</a> Journal of histotechnology. 35, 63-67 (2012).</li><li>Gibson-Corley, K. N., Olivier, A. K., Meyerholz, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+for+valid+histopathologic+scoring+in+research.">Principles for valid histopathologic scoring in research.</a> Veterinary pathology. 50, 1007-1015 (2013).</li><li>Stolfi, C., 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=Involvement+of+interleukin-21+in+the+regulation+of+colitis-associated+colon+cancer.">Involvement of interleukin-21 in the regulation of colitis-associated colon cancer.</a> The Journal of experimental medicine. 208, 2279-2290 (2011).</li><li>Begley, C. G., Ellis, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+development:+Raise+standards+for+preclinical+cancer+research.">Drug development: Raise standards for preclinical cancer research.</a> Nature. 483, 531-533 (2012).</li><li>Peters, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Practical Guide to Frozen Section Technique. , (2010).</li><li>Rosai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rosai and Ackerman's Surgical Pathology. , (2011).</li><li>Blumberg, R. S., Saubermann, L. J., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+mucosal+inflammation+and+their+relation+to+human+inflammatory+bowel+disease.">Animal models of mucosal inflammation and their relation to human inflammatory bowel disease.</a> Current opinion in immunology. 11, 648-656 (1999).</li><li>Wirtz, S., Neufert, C., Weigmann, B., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+induced+mouse+models+of+intestinal+inflammation.">Chemically induced mouse models of intestinal inflammation.</a> Nature. 2, 541-546 (2007).</li><li>Kaser, A., Zeissig, S., Blumberg, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease.">Inflammatory bowel disease.</a> Annual review of immunology. 28, 573-621 (2010).</li><li>Cribi&#249;, F. M., Burrello, C., 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=Implementation+of+an+automated+inclusion+system+for+the+histological+analysis+of+murine+tissue+samples:+A+feasibility+study+in+DSS-induced+chronic+colitis.">Implementation of an automated inclusion system for the histological analysis of murine tissue samples: A feasibility study in DSS-induced chronic colitis.</a> European Journal of Inflammation. 16, 1-12 (2018).</li></ol>

We utilize different automated steps during the preparation of murine tissues for histopathologic analysis. This protocol aims at providing technical hints to increase the reproducibility and the standardization of the whole process, thus enhancing the overall quality of the final histopathological evaluation. We implemented automated instruments and methods for the preparation and embedding of tissues, routinely used in pathology core facilities for the study of human specimens.
To demonstrat...

In the last decades, several experimental models have been developed to dissect the pathogenic mechanisms leading to human diseases1,2. In order to assess the severity of a disease, researchers must evaluate the effect of a treatment and study the cytological and histological architectural variations or the amount of inflammation3. To perform on those experimental models, detailed histopathological analyses are needed, often comparing murine and human samples4,5.
Additionally, human samples are commonly processed and scored by histopathology core facilities and experienced human pathologists through standardized histopathological criteria and methods. Conversely, murine tissues are usually fixed, embedded and analyzed by researchers with limited experience of histopathological protocols. The quality and reliability of histopathological examination begins with the preparation of high-quality tissue sections. Several factors critically contribute to increase or decrease the quality of the final analysis, including fixation, macroscopic sectioning, processing, paraffin embedding, and embedding of the samples6,7.
All these passages involving manipulation of the sample are subjected to manual errors, including manual embedding of the samples and, to a lesser extent, manual microtome sectioning and staining. At present, the whole process of murine tissue preparation for histological evaluation relies on protocols that vary from laboratory to laboratory and manual protocols. The goal of this study is to implement standardized automated protocols to reduce errors and variability in murine histopathological examination.
To our knowledge, we describe here the first protocols for fully automated tissue processing and embedding for the histological evaluation of murine tissues; these are routinely used in pathology units for the analyses of human specimens. As a practical example of the feasibility of the method, a murine model of intestinal inflammation has been analyzed, i.e., the chronic colitis model caused by repeated administration of dextran sodium sulphate (DSS) in the drinking water8,9. This experimental setting closely resembles human inflammatory bowel diseases (IBD)10 since DSS-treated animals exhibit signs of intestinal inflammation, e.g., weight loss, loose stool or diarrhea, and shortening of the colon as well as fibrosis8,9,11. As observed for human IBD patients, DSS treatment generates a complex disease course. In this context, elaborate histological evaluations are required to understand the profound alteration of the tissue architecture. Thus, the implementation of the described protocols for increasing sample preparation quality might benefit researchers relying on the interpretation of histological and immunohistochemical analyses for murine experimental settings. Murine experimental models of human diseases involving alterations of the tissue architecture, the presence of cellular tissue infiltrate or inflammation in different tissues and organs (intestine, brain, liver, skin) could use the increased quality of the sample preparation for histopathological examination.

<table>
	<tbody>
		<tr>
			<td>Absolute Ethanol anhydrous</td>
			<td>Carlo Erba</td>
			<td>414605</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Absolute ETOH</td>
			<td>Honeywell</td>
			<td>02860-1L</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Aluminium Potassium Sulfate</td>
			<td>SIGMA</td>
			<td>A6435</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Aniline Blue</td>
			<td>SIGMA</td>
			<td>415049</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>carbol Fuchsin</td>
			<td>SIGMA</td>
			<td>C4165</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>CD11b (clone M1/70)</td>
			<td>TONBO biosciences</td>
			<td>35-0112-U100</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>CD20 IHC (clone SA275A11)</td>
			<td>Biolegend</td>
			<td>150403</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>CD3 (17A2)</td>
			<td>TONBO biosciences</td>
			<td>35-0032-U100</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>CD4 (GK1.5)</td>
			<td>BD Biosciences</td>
			<td>552051</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>CD45.2 (clone 104)</td>
			<td>BioLegend</td>
			<td>109837</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>CD8 (53-6.7)</td>
			<td>BD Biosciences</td>
			<td>553031</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Citrate Buffer pH 6 10X</td>
			<td>SIGMA</td>
			<td>C9999</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Dab</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>DPBS 1X</td>
			<td>Microgem</td>
			<td>L0615-500</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>DSS</td>
			<td>TdB Consultancy</td>
			<td>DB001</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA</td>
			<td>E9884</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>EnVision Flex Peroxidase-Blocking Reagent</td>
			<td>DAKO</td>
			<td></td>
			<td>compreso in GV80011-2</td>
		</tr>
		<tr>
			<td>EnVision Flex Substrate</td>
			<td>DAKO</td>
			<td></td>
			<td>compreso in GV80011-2</td>
		</tr>
		<tr>
			<td>EnVision Flex/HRP</td>
			<td>DAKO</td>
			<td></td>
			<td>compreso in GV80011-2</td>
		</tr>
		<tr>
			<td>EnVision Flex+ Rat Linker</td>
			<td>DAKO</td>
			<td></td>
			<td>compreso in GV80011-2</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>VWR</td>
			<td>1.09844</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>F4/80 (clone BM8)</td>
			<td>BioLegend</td>
			<td>123108</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>PanReac</td>
			<td>2,529,311,215</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>glacial acetic acid</td>
			<td>SIGMA</td>
			<td>71251</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Goat-anti-Rat-HRP</td>
			<td>Agilent DAKO</td>
			<td>P0448</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Haematoxylin</td>
			<td>DIAPATH</td>
			<td>C0303</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>LEICA Rotary microtome (RM2255)</td>
			<td>Leica</td>
			<td>RM2255</td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>Ly6g (clone 1A8)</td>
			<td>BD Biosciences</td>
			<td>551459</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Mercury II Oxide</td>
			<td>SIGMA</td>
			<td>203793</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Omnis Clearify Clearing Agent</td>
			<td>DAKO</td>
			<td>CACLEGAL</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Omnis EnVision Flex TRS</td>
			<td>DAKO</td>
			<td>GV80011-2</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>SIGMA</td>
			<td>O3756</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sakura</td>
			<td>7052</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>Peloris</td>
			<td>LEICA</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>SIGMA</td>
			<td>P4937</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>RPMI 1640 without L-Glutamine</td>
			<td>Microgem</td>
			<td>L0501-500</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>STS020</td>
			<td>Leica</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>Tissue-Teck Paraform Sectionable Cassette</td>
			<td>SAKURA</td>
			<td>7022</td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>Tissue-Tek Automated paraffin embedder</td>
			<td>Sakura</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>J.T.Baker</td>
			<td>8080.1000</td>
			<td>reagent</td>
		</tr>
	</tbody>
</table>

using robotic systems to process and embed colonic murine samples for histological analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Experimental chronic colitis induced by repeated administration of DSS in the drinking water is a murine model of intestinal inflammation closely resembling human IBD8,9. Figure 1 describes the effects of DSS treatment, including colon shortening (Figure 1A), a widely-used parameter to score the presence of DSS-induced inflammation, and colonic expression of pro-inflammat...

Watch this Scientific Journal Video about Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses at JoVE.com

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

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