Name: performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification
Uploaded: 2012-09-15T14:00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification at JoVE.com

We would like to thank Marc Leonard from Carleton Immersive Media Studio for expert technical assistance in video production and editing and Dr. Martin Bertrand from Carleton Immersive Media Studio / Neural Regeneration Laboratory for visual model assistance. The authors gratefully acknowledge the expert advice of Dr. Marilyn Keaney and all of her dedicated staff at the University of Ottawa Animal Care and Veterinary Services. This work was funded by the Canadian Institute of Health Research (CIHR, MOP 62826) to SALB, the CIHR Institute of Aging and Strategic Training Initiative in Health Research/CIHR Training Program in Neurodegenerative Lipidomics (TGF 96121) to SALB and SF, Canadian Foundation for Innovation to SF, Ontario Innovation Trust to SF, and Autodesk Research to SF. ACM receives a CIHR Banting and Best doctoral award. NV receives a post-professional fellowship from Institute of Aging and CIHR Training Program in Neurodegenerative Lipidomics.

1. Preparing Reagents 
<ol>
 <li>For sterile vaginal lavage, autoclave double distilled water (ddH2O) and store in a tightly sealed container at room temperature until needed. </li>
 <li>For cytological assessment, add 0.1 g of crystal violet powder to 100 ml of ddH2O. Mix well. Crystal violet stain (0.1%) can be stored in a tightly sealed container at room temperature until needed. </li>
</ol>
2. Collecting Vaginal Cells (Vaginal Lavage)
<ol>
 <li>Place a latex bulb on the end of a sterile 200 &mu;l tip and draw up approximately 100 &mu;l of sterile ddH2O using the gradations on the tip as a volume guideline. </li>
 <li>Lift the mouse out of her cage and place her on the cage hopper (lid) with her hind/rear end towards you. </li>
 <li>Firmly grasp the tail and elevate the rear end. The mouse will now have only her front paws grasping the hopper. At this point the mouse may urinate. If so, wait until urination stops. Should there be urine left at the entrance to the vaginal canal, you may want to rinse the opening with excess ddH2O using a separate tip (i.e., not your sample collection tip).</li>
 <li>Place the end of the ddH2O-filled tip at the opening of the vaginal canal taking care to not penetrate the orifice as vaginal (and cervical) stimulation can induce pseudopregnancy in rats1,2. Recent reports suggest mice are less susceptible to this effect nonetheless care should be taken to minimize the degree of invasiveness in repeated analyses3. </li>
 <li>Gently depress the bulb to expel a quarter to half of the volume of water (~25-50 &mu;l) at the opening of vaginal canal. The liquid will spontaneously aspirate into the canal without tip insertion. Slowly release the pressure exerted on the bulb. The fluid will withdraw back into the tip. Avoid releasing pressure too quickly to prevent aspiration of fluid into the bulb. A filtered tip may be useful for this purpose. </li>
 <li>Repeat the previous step 4-5 times using the same tip, bulb, and fluid to obtain a sufficient number of cells in a single sample. </li>
 <li>Place the fluid on glass slide, and allow the smear to completely dry at room temperature. Once dry, these estrous smears can be stained immediately or stored and stained at a later date. </li>
</ol>
3. Cytological Staining using Crystal Violet*4
<ol>
 <li>Place the dry slide in a coplin jar (or other comparable staining vessel) containing the crystal violet stain for 1 min. </li>
 <li>Remove to a second coplin jar containing ddH2O. Wash the slide with ddH2O for 1 min. Repeat. </li>
 <li>Remove the excess ddH2O from the edges of the slide with a light-duty tissue wiper, avoiding contact with the stained smear.</li>
 <li>Pipette approximately 15 &mu;l of glycerol on top of the smear and coverslip. Alternatively, other histological mounting reagents can be utilized to obtain a more permanent, non-diffusing stain. </li>
</ol>
 * The staining method described here is the simplest procedure that can be performed in any laboratory. Other methods can provide additional details. For example, using Papanicolaou staining, the maturity of nucleated epithelial cells can be distinguished with less mature cells stained turquoise and more mature cells pink- or orange-stained. These differences can be used to stage early or late proestrus.4 
4. Vaginal Cytology
<ol>
 <li>Examine the smear under light microscopy to determine cell types present. Microscopic examination should be done immediately after staining as the crystal violet will diffuse from the cells over time when using glycerol for coverslipping. Photomicrographs should be taken at time of analysis to document cytology.</li>
 <li>Start by examining the entire smear at a lower magnification. Select a representative area and move to a higher magnification. You will see cornified squamous epithelial cells, leukocytes, and/or nucleated epithelial cells (representative results, Figure 1A-C). The ratio of cells present will allow you to determine the estrous stage of your mouse at time of sample collection (representative results, Figure 1D-G) and her immediate hormonal status (discussion, Figure 2). </li>
</ol>
5. Representative Results
Cytology: Three primary cell types can be detected in vaginal smear samples: (1) nucleated epithelial cells (Figure 1A), (2) cornified squamous epithelial cells (Figure 1B), and (3) leukocytes (Figure 1C). Nucleated epithelial cells have a lightly stained cytoplasm, darker stained plasma membrane, and an oval nucleus (Figure 1A). Cornified squamous epithelial cells are uniformly stained, more polygonal in shape than their nucleated epithelial predecessors, and lack a nucleus (Figure 1B). Polymorphonuclear leukocytes can be distinguished from epithelial cells by their irregular shape, darkly stained polymorphic nuclei, and small size (Figure 1C, black arrows). Should urine contamination be present in the smear, uric acid crystals are readily detected by their crystalline structures dissimilar to any expected cell types (Figure 3). Should this occur, and obscure detection of predominant cell type, the smear should be discarded and not used for staging purposes.
Staging: The relative ratio of cell types observed in smears can be used to identify the stage of the estrous cycle of your mouse on the day of sample collection (Figure 1D-G). During proestrus, cells are almost exclusively clusters of round, well-formed nucleated epithelial cells (Figure 1D, representative cell indicated by white arrow). During estrus, cells are predominantly cornified squamous epithelial cells, present in densely packed clusters (Figure 1E, representative cell indicated by arrowhead). During metestrus, small darkly stained leukocytes predominate (Figure 1F, representative cell indicated by black arrow). Cornified squamous epithelial cells may be observed, often in fragments, (Figure 1F, representative cell indicated by black arrowhead). During diestrus, rare cornified squamous epithelial cells may still be present (Figure 1G, representative cell indicated by black arrowhead), however leukocytes still predominate (Figure 1G, representative cell indicated by black arrow). Metestrus can be distinguished from diestrus by the appearance of nucleated epithelial cells in diestrus (Figure 1G, representative cell indicated by white arrow). 
<img src="/files/ftp_upload/4389/4389fig1.jpg" alt="Figure 1" /> 
Figure 1. Cytological assessment of vaginal smears can be used to identify estrous stage. Three main cell types are detected in vaginal smear samples: (A) nucleated epithelial cells, (B) cornified squamous epithelial cells, and (C) leukocytes. The ratio of these cell types present in the smear can be used to identify mice in (D) proestrus, (E) estrus, (F) metestrus, or (G) diestrus as described in representative results. Black arrowheads in E, F and G point to representative cornified squamous epithelial cells. Black arrows in C, F and G point to representative leykocytes. White arrows in D and G highlight representative nucleated epithelial cells.
<img src="/files/ftp_upload/4389/4389fig2.jpg" alt="Figure 2" /> 
Figure 2. Vaginal smear cytology reflects underlying endocrine events. Details are also provided in Discussion. <a href="https://www.jove.com/files/ftp_upload/4389/4389fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/4389/4389fig3.jpg" alt="Figure 3" /> 
Figure 3. Uric acid crystals may be present following crystal violet staining of urine-contaminated samples. (A) Crystals are transparent and can be of various sizes (arrows and boxed region magnified in (B)). No cells are present in this field. Should uric acid crystal contamination within fields used for cytological staining be present, it may be difficult to accurately identify cell types present and the smear should be discarded. Scale bars = 50 &mu;m.

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The authors declare no conflict of interest. All experiments on animals were performed in strict accordance with the guidelines and regulations set forth by the University of Ottawa Animal Care Committee and the Canadian Council on Animal Care.

These changes in cell typology are indicative of underlying endocrine events. The proestrus phase of the estrous cycle corresponds to the human follicular phase of the menstrual cycle5 and is defined by a pre-ovulatory increase in circulating 17-&beta;-estradiol levels6, as well as a small surge in prolactin7 (Figure 2, Proestrus, left panel). The increase in 17-&beta;-estradiol indirectly stimulates gonadotropin-releasing hormone neurons in the hypothalamus and septum that, in turn, activate responsive cells in the anterior pituitary to release luteinizing hormone and follicle-stimulating hormone into the circulation8,9 (Figure 2, Proestrus, left panel). In vaginal smears taken from animals in proestrus, cells are almost exclusively oval nucleated epithelial cells (Figure 1D, Figure 2, Proestrus, right panel). The peak in follicle-stimulating hormone levels signals ovulation and entry into estrus10,11. During estrus, 17-&beta;-estradiol levels decline and prolactin levels peak6,7 (Figure 2, Estrus, left panel). Vaginal smears are characterized by almost exclusive detection of irregular-shaped cornified squamous epithelial cells often in clumps (Figure 1E, Figure 2, Estrus, right panel). Entry into metestrus coincides with a continuous rise in progesterone hormone levels6 and corresponds to the beginning of human luteal phase12 (Figure 2, Metestrus, left panel). As progesterone levels start to rise and there is a small surge in 17-&beta;-estradiol levels in response to corpus luteum activation6,13,14 (Figure 2, Metestrus, left panel). The cell types present in vaginal smears during this stage are fragmented, cornified epithelial cells and smaller darker stained leukocytes (Figure 1F, Figure 2, Metestrus, right panel). Finally, entry into diestrus in mice occurs and circulating progesterone levels peak6, corresponding to the human late luteal phase12. Regression of the corpus luteum leads to a subsequent sharp decline in progesterone levels15,16 (Figure 2, Diestrus, left panel). Leukocytes predominate in smears during diestrus. The frequency of cornified epithelial cells is reduced and nucleated epithelial cells begin to be detected just prior to transition to proestrus (Figure 1G, Figure 2, Diestrus, right panel). 
In summary, this simple, routine protocol can be used to estimate daily hormonal fluctuations and establish estrous stage in experimental mice without altering reproductive status if the following precautions are taken. Sampling should be performed no more than once daily using the non-invasive protocol described here as compared to repeated penetration of the vaginal canal, aspiration, and agitation. This can cause vaginal irritation resulting in an inflammatory response17 resulting in leukocytes and other cell types to be present in smears that may confound cytological assessment. Moreover, even in colony-housed females, it is normal to see extended diestrus and estrus stages in different mice as well induction of anestrous18 and this identification is useful in the interpretation of hormonal impact in reproductive, gender, and disease studies. Variability in cycle length is also introduced with age and by housing differences within colonies (individual or group-housing) of females7,19-21. Females housed in female-only colonies can cease cycling and enter a state of prolonged diestrus18,22,23 although cycling can be re-instated by exposure to cages pretreated with male urine to elicit cycling24,25. Thus, to establish individual cycle lengths for a given mouse, it is recommended that the non-invasive assessments described here be performed daily, with care, until two complete cycles are observed.

<table border="1">
	<tbody>
 	<tr>
 	<td>Name</td>
 <td>Company</td>
 <td>Catalogue #</td>
 </tr>
 <tr>
 	<td>Sterile 200 &mu;l pipette tips</td>
 <td>Diamed</td>
 <td>E340901</td>
 </tr>
 <tr>
 	<td>Latex bulb (1 ml)</td>
 <td>Fisher</td>
 <td>03-488-21</td>
 </tr>
 <tr>
 	<td>Glass microscope slides</td>
 <td>Fisher</td>
 <td>12-550-15</td>
 </tr>
 <tr>
 	<td>Crystal Violet stain (25 g)</td>
 <td>Fisher</td>
 <td>C581-25</td>
 </tr>
 <tr>
 	<td>Light-duty Tissue Wipers</td>
 <td>VWR</td>
 <td>82003-820</td>
 </tr>
 <tr>
 	<td>Glycerol</td>
 <td>Fisher</td>
 <td>BP229-1</td>
 </tr>
 <tr>
 	<td>Microscope Cover Glass (22x30)</td>
 <td>Fisher</td>
 <td>12-544A</td>
 </tr>
 </tbody>
</table>

performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Watch this Scientific Journal Video about Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification at JoVE.com

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

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A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

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Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...