We thank Prof. Weiguo Zou and S. Kato for reagents and mice and the members of the Zou laboratory for useful discussions. We also thank the Laboratory for Digitized Stomatology and Research Center for Craniofacial Anomalies of Shanghai Ninth People&#39;s Hospital for assistance. This work was supported in part by grants from the National Natural Science Foundation of China (NSFC) [81570950,81870740,81800949], Shanghai Summit &#38; Plateau Disciplines, the SHIPM-mu fund from the Shanghai Institute of Precision Medicine, Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine [JC201809], the Incentive Project of High-Level Innovation Team for Shanghai Jiao Tong University School of Medicine, the Cross-disciplinary Research Fund of Shanghai Ninth People&#39;s Hospital, Shanghai JiaoTong university School of Medicine [JYJC201902]. And L.J. is a scholar of the Outstanding Youth Medical Talents, Shanghai &#34;Rising Stars of Medical Talent&#34; Youth Development Program and the &#8220;Chen Xing&#8221; project from Shanghai Jiaotong University.

All methods relating to the animals described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiaotong University School of Medicine.
1. Breeding of osteoclast specific Stat3 deletion mice
NOTE: Stat3fl/fl mice were obtained commercially. Ctsk-Cre mice were provided by S. Kato (University of Tokyo, Tokyo, Japan12). The mice were bred and maintained under sp...

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Technical considerations of endogenous phosphatase activity.</a> American Journal of Clinical Pathology. 84 (4), 476-480 (1985).</li><li>Solberg, L. 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=Increased+tartrate-resistant+Acid+phosphatase+expression+in+osteoblasts+and+osteocytes+in+experimental+osteoporosis+in+rats.">Increased tartrate-resistant Acid phosphatase expression in osteoblasts and osteocytes in experimental osteoporosis in rats.</a> Calcified Tissue International. 94 (5), 510-521 (2014).</li><li>Tambutt&#233;, 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=Calcein+labelling+and+electrophysiology:+insights+on+coral+tissue+permeability+and+calcification.">Calcein labelling and electrophysiology: insights on coral tissue permeability and calcification.</a> Proceedings. 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The authors have nothing to disclose.

Genetically engineered mouse models are commonly used for studying the mechanism and pharmaceutical treatment of human disease13. Ctsk-Cre mice have been widely used for functional studies of osteoclasts6. The present study described the protocols of the methods to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.
Histological analysis is the best intuitive method to detect bone metabolis...

Skeletal bone is the main load-bearing organ of the human body and is under pressure from both the internal and external environment during walking and exercise1. Throughout one&#8217;s life, bones continuously go through self-renewal, which is balanced by osteoblasts and osteoclasts. The process of osteoclasts clearing old bones and osteoblasts forming new bone maintains the homeostasis and mechanical function of the skeletal system2. Disturbance in the balance may induce bone metabolic diseases, such as osteoporosis. Osteoporosis, which is caused by excess osteoclastic activity, is globally prevalent and causes substantial economic losses to society2,3,4. According to the limited number of drugs available for osteoporosis treatment and their risk of adverse effects4, it is important to unveil the details of osteoclast formation and activity.
Osteoclasts derived from the monocyte/macrophage hematopoietic lineage have multiple nuclei (may have 2 to 50 nuclei) and are large (usually greater than 100 &#956;m in diameter)2. Although the exploration of mechanisms and the screening of drugs for osteoclastic disorders have been widely improved via in vitro osteoclast culture, the complicated organic reactions make in vivo evidence indispensable for the targeted therapy. Due to genetic and pathophysiological similarities between mice and humans, genetically engineered mouse models are commonly used for studying the mechanisms and the pharmaceutical treatments of human disease in vivo6. The Cre-loxP system is a widely-used technology for mouse gene editing and has enabled researchers to investigate gene functions in a tissue-/cell-specific manner5. Cathepsin K (CSTK) is a cysteine protease secreted by osteoclasts that can degrade bone collagen8. It is well accepted that CTSK is selectively expressed in mature osteoclasts; therefore, Ctsk-Cre mice are considered to be a useful tool for functional studies of osteoclasts and has been used6.
The signal transducer and activator of transcription (STAT) family is classical and highly significant in immunity and cancer progression and development7,8. Among seven STATs, STAT3 is reported to be the most relevant to bone homeostasis9,10. Several in vivo studies have reported that specific inactivation of STAT3 in osteoblasts decreases bone formation9,10. Nevertheless, solid evidence regarding the participation of STAT3 in osteoclast formation and bone metabolism in vivo is still limited. Recently, we provided in vivo evidence with an osteoclast-specific Stat3 deletion mouse model (Stat3fl/fl; Ctsk-Cre, hereafter called Stat3Ctsk) that STAT3 participates in osteoclast differentiation and bone metabolism11. In the present study, we describe the methods and protocols that we used to analyze the changes in bone mass, bone histomorphology, and bone anabolism and catabolism of the Stat3Ctsk mice in order to study the influence of osteoclast-specific STAT3 deletion on bone homeostasis.

<table>
	<tbody>
		<tr>
			<td>4% Paraformaldehyde solution</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E672002</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>80000360</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia solution</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-Cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>Electron Microscopy Sciences</td>
			<td>13710</td>
			<td></td>
		</tr>
		<tr>
			<td>DeCa RapidlyDecalcifier</td>
			<td>Pro-Cure</td>
			<td>DX1100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>Electron Microscopy Sciences</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBED 812 RESIN</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Beyotime Biotechanology</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Scanco Medical AG</td>
			<td>&#956;CT 80</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>10018918</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E675007</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>Electron Microscopy Sciences</td>
			<td>19000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary microtome</td>
			<td>Leica</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Stat3fl/fl mice</td>
			<td>GemPharmatech Co., Ltd</td>
			<td>D000527</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

skeletal phenotype analysis of a conditional stat3 deletion mouse model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Using the present protocol, osteoclast specific Stat3 deletion mice were generated to study the influence of STAT3 deletion on osteoclast differentiation. Stat3Ctsk mice and their wildtype (WT) littermates were bred and kept after genotyping. Bone marrow macrophages were isolated and cultured into osteoclasts, and STAT3 deletion in Stat3Ctsk mice was demonstrated (Figure 1).
Femora reconstruction a...

Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

Genetically engineered mouse models are powerful tools for studying the mechanisms of human disease in vivo. Analyzing the skeletal phenotype of mice is the basis of skeletal research. This particle describes some typical techniques for analyzing the skeletal phenotype which may be interesting for those who are new to skeletal tissue research.When attempting this particle keep in mind that animal experiments take time, so collect as many high quality samples as possible during each experiment, even if you don't need them in the short term. Begin by placing a euthanized mouse in a supine position and gently dislocating the bilateral hip joints by hand. Use ophthalmic scissors to vertically cut off the skin from the distal tibia and then remove all skin from the hind limb.Cut off the articular ligament of the right hip joint and knee joint with scissors to separate the hind limb. Then cut the bone at both ends to fully immerse the bone in 4%PFA. Cut the trochanter and the junction of the fibula.Immerse the hind limb in 4%PFA, keeping the right hind limb for paraffin sectioning. Cut the articular ligaments of the left hip and knee joints with scissors and gently remove the soft tissue. Separate the tibia and femur and immerse them separately in 75%ethanol.Keep the femora for micro CT scanning and the tibiae for calcein and alizarin red double labeling. To prepare the paraffin sections, gently wash the fixed right hind limb three times with PBS for 10 minutes per wash. Then decalcify it in 15%EDTA with an ultrasonic decalcifier for three to four weeks until the bones can be bent, replacing the decalcifying fluid every other day.After decalcification, wash the specimens three times with PBS and immerse them in 75%ethanol at four degrees Celsius overnight. On the second day, sequentially immerse specimens in 95%ethanol, 100%ethanol and xylene for one hour each. Immerse the specimens in half xylene and half paraffin for 30 minutes, then in paraffin at 65 degrees Celsius overnight.To embed the specimen, submerge it in paraffin, placing the femur and tibia at a 90 degree angle. When the paraffin has fully cooled, remove the specimens from the embedding tank. Number and store them at negative 20 degrees Celsius overnight.Bake the paraffin sections at 65 degrees Celsius for 30 minutes, then de wax them by immersing them in xylene for 10 minutes. Immerse the sections three times with fresh xylene each time. Rehydrate the sections by immersing them sequentially in 100%ethanol, 95%ethanol, 70%ethanol and distilled water for five minutes each.Prepare the staining solution using the TRAP staining kit and warm it to 37 degrees Celsius. Add 50 to 100 microliters of staining solution to each section and incubate them in a 37 degrees Celsius humid chamber for 20 to 30 minutes. Check the staining status of the osteoclasts under a light microscope every five minutes until red multinucleated osteoclasts can be seen.Then end the reaction with water. Counterstain the sections in hematoxylin solution for 30 seconds and create a stable blue color by immersing them in 1%ammonia solution for one minute. Then rinse them in slowly running tap water.Mount the sections using cover slips with neutral balsam and dry them overnight. Capture three to five fields of view with a microscope and analyze the trabecular perimeter with image J.Use the straight line tool to measure the length of the scale bar as L1.Then use the segmented line tool to measure the length of the trabecular perimeter as L2.Calculate the physical length and count the number of TRAP positive cells with more than three nuclei. After fixation, gently wash the tibiae three times with PBS and sequentially immerse the specimens in 95%ethanol, 100%ethanol and xylene for five minutes each.Immerse the specimens in acetone for 12 hours and half acetone and half resin for two hours, and in pure resin in a drying oven overnight. Add pure resin into a suitable silica gel embedding tank and place the specimens in the tank avoiding bubbles. Polymerize the resin in a drying oven at 60 degrees Celsius for 48 hours.Cut the specimens into five micrometer thick sections continuously with a rotary microtome and store the rest of the samples with desiccant at room temperature. Adhere the sections with tweezers in a drop of 75%ethanol and mount them with cover slips using neutral balsam. Capture the red and green fluorescence labeling with a fluorescence microscope.Osteoclasts-specific Stat3 deletion mice were generated to study the influence of Stat3 deletion on osteoclast differentiation. Femoral reconstruction and quantitative analysis by micro CT indicated that the bone mass of the Stat3 Ctsk mice was increased compared to wild type mice. Histo morphology of the femora from wild type and Stat3 Ctsk mice was examined via H and E staining.Osteoclastogenic activity was detected using TRAP staining. Osteoclasts are large TRAP positive cells with multiple nuclei. The number of TRAP positive osteoclasts was lower in Stat3 Ctsk mice, compared with wild type mice, indicating that Stat3 deficiency impaired osteoclast formation.Osteogenesis was measured with calcein and alizarin red double labeling. The area between the calcein and the alizarin red fluorescence represents newly formed bone. The deleted Stat3 in osteoclasts did not influence bone anabolism.Sagittal paraffin sections in which the cartilage later were symmetrical and that showed that clear M-shaped line was used here. For the studies, we'll include more characteristics of skeletal system, such as mechanical properties.

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4.2K visualizações.  National Clinical Research center of Stomatology. Genetically engineered mouse models are powerful tools for studying the mechanisms of human disease in vivo. Analyzing the skeletal phenotype of mice is the basis of skeletal research. This particle describes some typical techniques for analyzing the skeletal phenotype which may be interesting for those who are new to skeletal tissue research.When attempting this particle keep in mind that animal experiments take time, so collect as many high quality samples as possible during each exp...