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 7 days to the treatment group and water to the control group. 
	NOTE: This model induces a chronic transmural intestinal inflammation9.</li>
	<li>After 7 days, stop DSS treatment and give water to both groups for 14 days. Repeat this process 3 times. 
	NOTE: Repetitive administration of DSS induces alterations of the colonic mucosa closely resembling human IBD, i.e., fibrosis9.</li>
	<li>Sacrifice mice according to the procedures authorized by the Institutional IACUC, i.e., by CO2 inhalation.</li>
	<li>Open the mouse abdomen and peritoneum with a scalpel. 
	NOTE: Sterility is recommended but not strictly required.</li>
	<li>Separate the colon from the small intestine with forceps and tweezers. Excise the colon with tweezers and forceps. 
	NOTE: Colon length measurement (Figure 1A) is a method to determine if colitis occurred in DSS-treated mice.</li>
	<li>Rinse the colon in a Petri dish with 10 mL of cold 1x phosphate buffer saline (PBS) and gently press to remove fecal material.</li>
</ol>
2. Murine Tissues Fixation
<ol>
	<li>Immerse each murine colon specimen in 10 mL of 10% Neutral Buffered Formalin (NBF) at RT (room temperature). 
	NOTE: Sterility is recommended but not strictly required.</li>
	<li>Fix the tissue for 18&#8211;24 h at RT.</li>
</ol>
3. Colon Sectioning and Tissue Preparation
<ol>
	<li>Remove fixed tissues from the NBF container with small tweezers and put them in a Petri dish. Put the colon on a sectioning work plate with small tweezers.</li>
	<li>Cut colons in fragments (0.2 cm to 0.3 cm lengths) with a sterile scalpel. This fragment length is optimal in order not to exceed the thickness of the cassette.</li>
	<li>Pick up one colon segment with small tweezers (Figure 2A). Insert one colon segment into one of the plastic protruding tips of the orientated paraffin embedding cassette with small tweezers (Figure 2B).</li>
	<li>Repeat the operation (3.2&#8211;3.3) with an additional 3 colon segments per cassette. Avoid inserting the segments into adjacent protruding tips, to minimize overlapping of the tissues</li>
	<li>Tightly close the cassette by carefully pushing the four edges (Figure 2C). Avoid squeezing the tissue to prevent tissue damage.</li>
	<li>Insert the grid into a plastic supporting frame. Label the supporting frame to identify the sample. Repeat for each biological sample.</li>
</ol>
4. Tissue Processing
<ol>
	<li>Turn on the automated processor by pushing the power button (Figure 3A, 3B).</li>
	<li>Warm up for 1 h to ensure paraffin wax melting. Wait until the instrument confirms the paraffin is completely melted, by observing the presence of the dedicated icon (Figure 3C).</li>
	<li>Insert each oriented cassette (containing the tissue specimens) manually into the metal basket provided by the automated processor (Figure 3D). Place the cassettes vertically by lining them close to one to the other to optimize the basket occupancy.</li>
	<li>Close the metal basket (Figure 3E).</li>
	<li>Open the lid of the retort (Figure 3F). The retort is the place where the basket is inserted into the machine. Insert the basket into the dedicated housing of the processor (Figure 3G). Close the lid of the retort (Figure 3H).</li>
	<li>Use the touch screen on the instrument computer to define the working protocol (Figure 3I). Choose the sequence of solutions, timing and temperature to be implemented according to the scheme provided in Table 1.</li>
	<li>Assign the protocol to be run on the retort containing the basket by clicking on the dedicated computer icon (Figure 3J). Start the protocol by clicking on the Start Button (Figure 3L).</li>
	<li>Wait until the instrument confirms the end of the protocol, by observing the presence of the dedicated icon and by hearing the alarm tone coming from the machine.</li>
	<li>Open the retort lid. Remove the basket from the processor (Figure 3M).</li>
</ol>
5.&#160; Tissue Embedding
<ol>
	<li>Turn on the automated embedder by pushing the power button (Figure 4A).</li>
	<li>Warm up for 1 h to ensure paraffin wax melting. Wait until the instrument confirms the paraffin is completely melted by observing the temperature of the paraffin bath indicated by the internal thermometer of the instrument.</li>
	<li>Manually transfer all the processed cassettes from the processor basket to the embedder rack. Each rack can contain up to 32 cassettes. (Figure 4B, 4C).</li>
	<li>Open the main embedder lid (Figure 4C, 4D, 4E).</li>
	<li>Use the touch screen on the instrument computer to signal to the robotic system that a rack is being inserted (Figure 4F).</li>
	<li>Open the inlet housing lid (Figure 4G). Insert the rack into the inlet housing. Each embedder can contain up to 4 racks simultaneously (Figure 4H, 4I). Close the inlet housing lid (Figure 4J).</li>
	<li>Use the touch screen on the instrument computer to start the embedding procedure, according to Table 2 (Figure 4K). 
	NOTE: Each rack of 32 cassettes takes 45 min to be embedded.</li>
	<li>Wait until the instrument confirms the end of the protocol, by observing the presence of the dedicated icon (Figure 4L).</li>
	<li>Remove the outlet rack (Figure 4M). Close the main embedder lid.</li>
	<li>Remove the embedded blocks (containing the orientation grids) from the rack. Transfer the embedded blocks in a storage cardboard box.</li>
</ol>
6. Micrometer Sectioning
<ol>
	<li>Turn on the cooling plate of the microtome. Set the temperature between -8 and -10 &#176;C.</li>
	<li>Turn on the thermostatic water bath of the microtome, containing 2 L of distilled water. Set the temperature of the water bath between 42&#8211;45 &#176;C.</li>
	<li>Place the paraffin embedded blocks on the cooling plate. Wait at least 5 min to allow the blocks to cool.</li>
	<li>Take one block from the cooling plate and place it into the microtome block holder.</li>
	<li>Set the microtome cut thickness to 10 &#181;m. Trim the block by cutting it 6 times at 10 &#181;m thickness.</li>
	<li>Change the thickness setting of the microtome from 10 &#181;m to 3 &#181;m. Cut one 3 &#181;m section for Hematoxylin and Eosin (H&#38;E) staining.</li>
	<li>Collect the 3 &#181;m section with a small brush. Put the 3 &#181;m section in the water bath, laying it carefully on the water surface to reduce tissue wrinkles. Collect the tissue section from the thermostatically controlled water bath on a single glass slide.
	<ol>
		<li>If needed, cut additional sections.</li>
	</ol>
	</li>
	<li>Label the glass slide by writing the sample identification.</li>
	<li>Put the slides in a 37 &#176;C oven for at least 10 min.</li>
	<li>Put glass slides into racks for immediate analyses or in storage boxes.</li>
</ol>
7. Hematoxylin and Eosin (H&#38;E) Staining
<ol>
	<li>Switch on the automated stainer by pushing the power button. Allow the initialization of the robotic arm, by observing the change of the loading bar on the instrument monitor.</li>
	<li>Insert the glass slides into the stainer rack, vertically inserting up to 30 slides.</li>
	<li>Label the rack with the appropriate radio frequency identification (RFID) plastic tag. RFID tagging is a system identifying the correct staining protocol loaded into the stainer computer.</li>
	<li>Insert the rack into the stainer. Allow the computer to automatically load and start the staining protocol according to the recognition of the RFID tag. 
	NOTE: The protocol for H&#38;E staining is described in Table 3.</li>
</ol>
8. Immunohistochemical Staining
<ol>
	<li>Perform immunohistochemical and Mallory trichrome staining as previously described7.</li>
</ol>

<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>

The authors have nothing to disclose.

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 demonstrate the potential applicability of this method, we chose a chronic murine experimental setting of intestinal inflammation, called DSS-induced chronic colitis model. This setting resembles the complex disease course observed in IBD patients, requiring elaborate histological evaluation to investigate the profound alterations of the tissue architecture occurring upon chronic intestinal inflammation. Altogether, the histological evaluation of these processes might greatly benefit from increased sample preparation quality. To note, this protocol can be applied to other murine tissues (i.e., spleens, lymph nodes, liver, brain), with the only difference being that the oriented cassette is not needed for tissues without a lumen.
The most important observation was that the decrease of manual errors, (i.e., the orientation of the sample) given the use of cassettes with grids and the elimination of cassette re-opening for the embedding (a step commonly performed during manual protocols), strongly enhanced the overall reproducibility of the analysis. With the protocol described here, the sample is manipulated only at the beginning of the preparation, when the experimenter inserts the tissue in the orientated cassettes. Once closed, the cassettes are never re-opened, thus ensuring the maintenance of the correct orientation and reducing manual errors3,11. The standardization of these two critical steps enhanced the whole quality of the subsequent analyses and decreased the number of lost or not assessable samples, problems both linked to the re-opening of the cassette or to the wrong orientation of the sample3,11.
We also succeeded in evaluating a complex biological event such as fibrosis (Figure 7), that is very often underestimated in murine models of experimental intestinal inflammation, by the implementation of the automated protocol. During the set-up of the protocol, we strictly standardized technical details such as the fixation time, which we realized must not exceed 24 h to avoid tissue alterations. By doing so, we could preserve the quality of subsequent immunohistochemical analyses.
The automated method improved the evaluation of those parameters associated to the alteration of the tissue architecture, especially in untreated mice. Indeed, the correct comparison of a pathological tissue with a healthy counterpart is critically important for the final assessment of the experimental model3.
Concerning the tissue processing, we tested different protocols to be run in the automated processor available in the laboratory. The protocol described here gave sound and highly consistent results. We also implemented a time length for the protocol. Since murine samples were comparable to human small biopsic specimens in size, a shorter protocol was sufficient to process murine (intestinal, in this case) tissues. Finally, the whole automated procedure reduced the total experimental time. From the beginning of the processing to the microtome cutting and slices preparation, we calculated that the total time required is 5 h. On the contrary, depending on the technical ability of the operator, the completion of the same protocols manually can require more than double the amount of time, especially during the processing and the embedding. One limit of the technique is that it requires automated processors and embedder to be performed to be in the laboratory.
In conclusion, we believe that the implementation of these automated protocols could greatly ameliorate the work of translational researchers dealing with murine experimental models of human diseases.

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/58654/using-robotic-systems-to-process-embed-colonic-murine-samples-for">Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses</a>.&#160; An author affiliation&#160;was updated.
The affiliation for Claudia&#160;Burrello and Federica Facciotti was updated from:
Department of Experimental Oncology, European Institute of Oncology
to:
Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/58654/using-robotic-systems-to-process-embed-colonic-murine-samples-for">Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses</a>.&#160; An author affiliation&#160;was updated.

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

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.

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 ...

using-robotic-systems-to-process-embed-colonic-murine-samples-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.0K Views.  Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico. Dit geautomatiseerde protocol maakt het mogelijk de histopathologische voorbereiding van murineweefsels met robotsystemen die routinematig worden gebruikt voor de verwerking en inbedding van menselijke monsters, waardoor de kwaliteit van de monsters sterk toeneemt. De belangrijkste voordelen van deze techniek zijn dat het aantal methodologische variabelen vermindert en de standaardisatie van procedurele stappen verbetert. Het demonstreren van de procedure, zal Francesca Boggio.Francesc...

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

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Medicine

Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations. To characterize proteome-level variation and tumor heterogeneity at scale, high-throughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&#38;E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

This protocol describes a high-throughput workflow for artificial intelligence-driven segmentation of pathology-confirmed regions of interest from stained, thin tissue section images for enrichment of histology-resolved cell populations using laser microdissection. This strategy includes a novel algorithm enabling the transfer of demarcations denoting cell populations of interest directly to laser microscopes.

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell ...

Cancer Research

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

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

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

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

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

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

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Biology

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion ...

Generating PDOX Mouse Model of Colorectal Cancer: Studying Progression of Colorectal Cancer in a Murine Model

Source: &#160;Moret, R. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59223"> Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.</a> J. Vis. Exp. (2019).
This video describes the protocol for generating a patient-derived orthotopic xenograft colorectal cancer mouse model to study the progression of colorectal cancer cells in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. This murine model helps in understanding primary tumor growth and metastases pattern.

Source: &#160;Moret, R. et al.  Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and ...

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

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