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...

An erratum was issued for: <a href="https://www.jove.com/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/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.

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 ...

This automated protocol allows the histopathological preparation of murine tissues with robotic systems routinely used for processing and embedding of human samples, greatly increasing the quality of the samples. The main advantages of this technique, are that it reduces the number of methodological variables and it improves the standardization of procedural steps. Demonstrating the procedure, will be Francesca Boggio.Francesca Boggio is a pathologist working in my laboratory. Begin by using small tweezers to transfer the fixed murine colon tissue samples from the neutral buffered formalin, onto a sectioning work plate in a Petri dish. Use a sterile scalpel to cut the colon into 0.2 to 0.3 centimeter fragments, and use the tweezers to place one segment into each of three non-adjacent plastic-protruding tips of an oriented paraffin-embedding cassette.Carefully push the four edges to close the cassette and insert the grid into a plastic supporting frame. Then, label the supporting frame to identify the sample and transfer the cassette into the grid. One hour before processing, turn the automated processor.When the instrument indicates that the paraffin has melted, manually insert the metal basket in the automated processor. When all of the cassettes have been inserted, close the basket and open the lid of the retort. Insert the basket into the dedicated housing of the processor and close the retort lid.Use the touchscreen on the instrument computer to define the working protocol selecting the sequence of solutions, timing, and temperature, to be implemented according to the scheme provided in the table. Click on the dedicated computer icon to assign the protocol to the retort containing the cassette basket, and click the start button. When the instrument confirms the end of the protocol with a dedicated icon and an alarm tone, open the retort lid and remove the basket from the processor.An hour before the tissue embedding, turn on the automated embedder and wait until the paraffin is completely melted as indicated by the paraffin bath thermometer on the instrument. Manually transfer up to 32 processed cassettes from the processor basket to the embedder rack and open the main embedder lid. Use the touchscreen to signal to the robotic system that a rack is being inserted and open the inlet housing lid.Insert up to four racks into the inlet housing and close the inlet housing lid. Then, use the touchscreen to start the embedding procedure as indicated in the table. When the instrument confirms the end of the protocol with the dedicated icon, remove the outlet rack and close the main embedder lid.Then, transfer the embedded blocks from the rack into a storage box. Colon shortening, and colonic expression of pro inflammatory genes, are widely used parameters for scoring the presence of dextran sodium sulfate or DSS induced inflammation. The infiltration of inflammatory cells into the intestinal lamina propria is also greatly enhanced by DSS treatment.H&E staining of untreated samples processed by automation as demonstrated, reveals fewer infiltrating inflammatory cells, epithelial alterations, and changes in mucosal architecture compared to samples from DSS treated animals. The quality of the murine tissue preparation and embedding performed by the automated instruments can be additionally confirmed by immunohistochemical staining for CD20 and Mallory Trichrome Staining. Notably, automated sample processing consistently allows the evaluation of a higher proportion of histological parameters, than do samples processed by the manual method.Take care to correctly insert the tissue into the grid to wait for the automated processor to be ready, and to wait for the paraffin to be completely melted. This method can be used to perform histological analysis of any murine tissue, with the qualitive of human histological samples which will be used in murine models of human pathologists. This technique has already been successful study for studying inflammatory tissue inflammation, and will be useful for studying models of colorectal murine cancer.As some reagents used in this protocol are hazardous, remember to always wear protective gloves, goggles and a lab coat when performing this procedure.

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

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...