<meta charset="utf8"></head><body>探索母体运动对大鼠后代健康的益处

<ol>
	<li>Hanson, M. A., Gluckman, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+developmental+conditioning+of+later+health+and+disease:+Physiology+or+pathophysiology.">Early developmental conditioning of later health and disease: Physiology or pathophysiology.</a> Physiol Rev. 94 (4), 1027-1076 (2014).</li><li>Calkins, K., Devaskar, S. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+origins+of+adult+disease.">Fetal origins of adult disease.</a> Curr Probl Pediatr Adolesc Health Care. 41 (6), 158-176 (2011).</li><li>Chavatte-Palmer, P., Tarrade, A., Rousseau-Ralliard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+before+and+during+pregnancy+and+offspring+health:+The+importance+of+animal+models+and+what+can+be+learned+from+them.">Diet before and during pregnancy and offspring health: The importance of animal models and what can be learned from them.</a> Int J Environ Res Public Health. 13 (6), 586 (2016).</li><li>Lapehn, S., Paquette, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placental+epigenome+as+a+molecular+link+between+prenatal+exposures+and+fetal+health+outcomes+through+the+dohad+hypothesis.">The placental epigenome as a molecular link between prenatal exposures and fetal health outcomes through the dohad hypothesis.</a> Curr Environ Health Rep. 9 (3), 490-501 (2022).</li><li>Shrestha, A., Prowak, M., Berlandi-Short, V. M., Garay, J., Ramalingam, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+obesity:+A+focus+on+maternal+interventions+to+improve+health+of+offspring.">Maternal obesity: A focus on maternal interventions to improve health of offspring.</a> Front Cardiovasc Med. 8, 696812 (2021).</li><li>Haire-Joshu, D., Tabak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+obesity+across+generations:+Evidence+for+early+life+intervention.">Preventing obesity across generations: Evidence for early life intervention.</a> Annu Rev Public Health. 37, 253-271 (2016).</li><li>Kusuyama, J., Alves-Wagner, A. B., Makarewicz, N. S., Goodyear, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+maternal+and+paternal+exercise+on+offspring+metabolism.">Effects of maternal and paternal exercise on offspring metabolism.</a> Nat Metab. 2 (9), 858-872 (2020).</li><li>Blaize, A. N., Pearson, K. J., Newcomer, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+maternal+exercise+during+pregnancy+on+offspring+chronic+disease+susceptibility.">Impact of maternal exercise during pregnancy on offspring chronic disease susceptibility.</a> Exerc Sport Sci Rev. 43 (4), 198-203 (2015).</li><li>Hinman, S. K., Smith, K. B., Quillen, D. M., Smith, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+in+pregnancy:+A+clinical+review.">Exercise in pregnancy: A clinical review.</a> Sports Health. 7 (6), 527-531 (2015).</li><li>Acog committee. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+activity+and+exercise+during+pregnancy+and+the+postpartum+period:+Acog+committee+opinion,+number+804.">Physical activity and exercise during pregnancy and the postpartum period: Acog committee opinion, number 804.</a> Obstet Gynecol. 135 (4), e178-e188 (2020).</li><li>Poole, D. 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=Guidelines+for+animal+exercise+and+training+protocols+for+cardiovascular+studies.">Guidelines for animal exercise and training protocols for cardiovascular studies.</a> Am J Physiol Heart Circ Physiol. 318 (5), H1100-H1138 (2020).</li><li>Beleza, J., 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=Self-paced+free-running+wheel+mimics+high-intensity+interval+training+impact+on+rats'+functional,+physiological,+biochemical,+and+morphological+features.">Self-paced free-running wheel mimics high-intensity interval training impact on rats' functional, physiological, biochemical, and morphological features.</a> Front Physiol. 10, 593 (2019).</li><li>August, P. M., Rodrigues, K. D. S., Klein, C. P., Dos Santos, B. G., Matt&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+gestational+exercise+practice+and+litter+size+reduction+on+maternal+care.">Influence of gestational exercise practice and litter size reduction on maternal care.</a> Neurosci Lett. 741, 135454 (2021).</li><li>Li, S., 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=Prenatal+exercise+reprograms+the+development+of+hypertension+progress+and+improves+vascular+health+in+shr+offspring.">Prenatal exercise reprograms the development of hypertension progress and improves vascular health in shr offspring.</a> Vascul Pharmacol. 139, 106885 (2021).</li><li>Li, S., 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=Exercise+during+pregnancy+enhances+vascular+function+via+epigenetic+repression+of+ca(v)1.2+channel+in+offspring+of+hypertensive+rats.">Exercise during pregnancy enhances vascular function via epigenetic repression of ca(v)1.2 channel in offspring of hypertensive rats.</a> Life Sci. 231, 116576 (2019).</li><li>Musial, B., 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=Exercise+alters+the+molecular+pathways+of+insulin+signaling+and+lipid+handling+in+maternal+tissues+of+obese+pregnant+mice.">Exercise alters the molecular pathways of insulin signaling and lipid handling in maternal tissues of obese pregnant mice.</a> Physiol Rep. 7 (16), e14202 (2019).</li><li>Dos Santos, A. S., 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=Resistance+exercise+was+safe+for+the+pregnancy+and+offspring's+development+and+partially+protected+rats+against+early+life+stress-induced+effects.">Resistance exercise was safe for the pregnancy and offspring's development and partially protected rats against early life stress-induced effects.</a> Behav Brain Res. 445, 114362 (2023).</li><li>Barnes, M. D., Heaton, T. L., Goates, M. C., Packer, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersystem+implications+of+the+developmental+origins+of+health+and+disease:+Advancing+health+promotion+in+the+21st+century.">Intersystem implications of the developmental origins of health and disease: Advancing health promotion in the 21st century.</a> Healthcare. 4 (3), 45 (2016).</li><li>Bakrania, B. A., George, E. M., Granger, J. P. <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+preeclampsia:+Investigating+pathophysiology+and+therapeutic+targets.">Animal models of preeclampsia: Investigating pathophysiology and therapeutic targets.</a> Am J Obstet Gynecol. 226, S973-S987 (2022).</li><li>Ferrari, 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=Exercise+during+pregnancy+and+its+impact+on+mothers+and+offspring+in+humans+and+mice.">Exercise during pregnancy and its impact on mothers and offspring in humans and mice.</a> J Dev Orig Health Dis. 9 (1), 63-76 (2018).</li><li>Lerman, L. O., 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=Animal+models+of+hypertension:+A+scientific+statement+from+the+american+heart+association.">Animal models of hypertension: A scientific statement from the american heart association.</a> Hypertension. 73 (6), e87-e120 (2019).</li><li>Mangwiro, Y. T. M., 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=Exercise+initiated+during+pregnancy+in+rats+born+growth+restricted+alters+placental+mtor+and+nutrient+transporter+expression.">Exercise initiated during pregnancy in rats born growth restricted alters placental mtor and nutrient transporter expression.</a> J Physiol. 597 (7), 1905-1918 (2019).</li><li>Kim, H., Lee, S. H., Kim, S. S., Yoo, J. H., Kim, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+maternal+treadmill+running+during+pregnancy+on+short-term+memory+and+hippocampal+cell+survival+in+rat+pups.">The influence of maternal treadmill running during pregnancy on short-term memory and hippocampal cell survival in rat pups.</a> Int J Dev Neurosci. 25 (4), 243-249 (2007).</li><li>Harris, J. E., 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=Exercise-induced+3'-sialyllactose+in+breast+milk+is+a+critical+mediator+to+improve+metabolic+health+and+cardiac+function+in+mouse+offspring.">Exercise-induced 3'-sialyllactose in breast milk is a critical mediator to improve metabolic health and cardiac function in mouse offspring.</a> Nat Metab. 2 (8), 678-687 (2020).</li><li>Zhang, Y., 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=Maternal+exercise+represses+nox4+via+sirt1+to+prevent+vascular+oxidative+stress+and+endothelial+dysfunction+in+shr+offspring.">Maternal exercise represses nox4 via sirt1 to prevent vascular oxidative stress and endothelial dysfunction in shr offspring.</a> Front Endocrinol. 14, 1219194 (2023).</li><li>Shan, M., 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=Maternal+exercise+upregulates+the+DNA+methylation+of+agtr1a+to+enhance+vascular+function+in+offspring+of+hypertensive+rats.">Maternal exercise upregulates the DNA methylation of agtr1a to enhance vascular function in offspring of hypertensive rats.</a> Hypertens Res. 46 (3), 654-666 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Caliper</td>
			<td>Mitutoyo Measuring Instruments (Shanghai) Co., Ltd</td>
			<td>530-101 N15</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Circular water bucket</td>
			<td>Naliya</td>
			<td>N/A</td>
			<td>Rat swimming</td>
		</tr>
		<tr>
			<td>Experimental Surgical Instrument&#160;</td>
			<td>Shenzhen RWD Life Technology Co., LTD</td>
			<td>SP0001-G</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Glass Microscope Slides</td>
			<td>Jiangsu Shitai experimental equipment Co., LTD</td>
			<td>80312-3161</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Glass petri dish</td>
			<td>Merck Life Technologies</td>
			<td>BR455751-10EA</td>
			<td>Offspring rearing</td>
		</tr>
		<tr>
			<td>Hairdryer</td>
			<td>Panasonic</td>
			<td>EH-WNE5H</td>
			<td>Post-swimming care</td>
		</tr>
		<tr>
			<td>Information Card</td>
			<td>Zhongke Life Science</td>
			<td>SS3</td>
			<td>Rat mating</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Shenzhen RWD Life Technology Co., LTD</td>
			<td>R510-22-10</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>IX71-F22PH</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Mating cage</td>
			<td>Zhongke Life Science</td>
			<td>SS3</td>
			<td>Rat mating</td>
		</tr>
		<tr>
			<td>Medical Absorbent Cotton</td>
			<td>Hongxiang Sanitary Materials Co., LTD</td>
			<td>N/A</td>
			<td>The pregnant rats are anticipating giving birth</td>
		</tr>
		<tr>
			<td>Rodent Anesthesia Machine, Gas Anesthesia</td>
			<td>Shenzhen RWD Life Technology Co., LTD</td>
			<td>R500IE</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Rodent breeding feed</td>
			<td>Beijing Huafukang Biotechnology Co., LTD</td>
			<td>1032</td>
			<td>Pregnant rat feeding</td>
		</tr>
		<tr>
			<td>Rodent maintenance feed</td>
			<td>Beijing Huafukang Biotechnology Co., LTD</td>
			<td>1022</td>
			<td>Offspring rearing</td>
		</tr>
		<tr>
			<td>Rodent sunflower seeds</td>
			<td>Jolly</td>
			<td>JP241</td>
			<td>Nutritional supplement</td>
		</tr>
		<tr>
			<td>Soundproof cotton</td>
			<td>Kufu Medical Instrument</td>
			<td>N/A</td>
			<td>The pregnant rats are anticipating giving birth</td>
		</tr>
		<tr>
			<td>Sterile Cotton Swab (2 mm diameter)</td>
			<td>Kufu Medical Instrument</td>
			<td>N/A</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Kufu Medical Instrument</td>
			<td>N/A</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Stroke-physiological Saline Solution (0.9% NaCl)</td>
			<td>Shandong Hualu Pharmaceutical Co., LTD</td>
			<td>N/A</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Beekman organism</td>
			<td>N/A</td>
			<td>The monitoring of rat swimming</td>
		</tr>
		<tr>
			<td>Towels</td>
			<td>Grace</td>
			<td>N/A</td>
			<td>Post-swimming care</td>
		</tr>
		<tr>
			<td>Transparent plastic container</td>
			<td>Naliya</td>
			<td>N/A</td>
			<td>Swimming adaptive training</td>
		</tr>
		<tr>
			<td>Ultraviolet light</td>
			<td>Merck Life Technologies</td>
			<td>Z169633-1EA</td>
			<td>Post-swimming care</td>
		</tr>
		<tr>
			<td>Water heater</td>
			<td>Haier</td>
			<td>EC6001-Q6S</td>
			<td>Rat swimming</td>
		</tr>
		<tr>
			<td>Weight Scale</td>
			<td>Electronlc Acale</td>
			<td>JM.A10001</td>
			<td>Body weight measurement</td>
		</tr>
		<tr>
			<td>Wistar Rats</td>
			<td>Vital river Laboratory Animal Technology Co., LTD</td>
			<td>N/A</td>
			<td>Experiment</td>
		</tr>
	</tbody>
</table>

swimming exercise protocol and care methods for pregnant rats

Read this Scientific Article Protocol about Author Spotlight: Enhancing Offspring Health with Maternal Exercise During Pregnancy at JoVE.com

<meta charset="utf8"></head><body>作者聚焦:怀孕期间通过母体运动增强大鼠的后代健康

怀孕大鼠的游泳锻炼方案和护理方法

The developmental origins of health and disease concept highlights the impact of early environments on chronic non-communicable diseases like diabetes, ...

swimming-exercise-protocol-and-care-methods-for-pregnant-rats

Research

JoVE Journal

Developmental Biology

Swimming Exercise Protocol and Care Methods for Pregnant Rats

607 次观看.  Beijing Sport University. 我们的研究描述了大鼠的繁殖方法。产前游泳训练产生大鼠和繁殖后护理方案，这将对运动生理学和围产期生物学的研究人员有很大帮助。随着怀孕的进展和大鼠腹部的肿胀，治疗和定期锻炼可能会导致腹部反复接触坚硬的表面。相比之下，游泳运动方案中的水环境提供浮力，降低了怀孕动物因运动而受伤的风险。母亲肥胖会导致后代心血管和...

视频: 作者聚焦:怀孕期间通过母体运动增强大鼠的后代健康

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium is known to affect bone metabolism. Here, we present a protocol aimed at investigating the effect of natural uranium exposure on the viability, the differentiation, and the function of osteoclasts, the cells in charge of bone resorption.

天然铀对体外破影响的分析方法

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

简化的3D 小脑鉴别协议, 可选2D 修改

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

使用定性和定量组织方法对小鼠胚胎先天性心脏缺陷的分析

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...