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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparing Animals for Experimental Studies
<ol>
	<li>Allow animals to acclimate to new housing and environment, to recover from potential physiologic effects of transport and handling. 
	Note: The following technique is applicable to all commercially available strains of mice. The number of animals to be used for an experiment is dependent on the study design and should be done with consultation from a statistician.</li>
</ol>
2. Identification and Isolation of Peritoneal Fat Depots
<ol>
	<li>Euthanize animals via CO2&#160;overdose&#160;and a secondary physical method to confirm death.&#160;</li>
	<li>As harvesting peritoneal fat depots constitutes internal organ removal (secondary method), make a midline incision close to the inguinal papillae through the peritoneal wall to expose the internal organs (Figure 1A).</li>
	<li>For the Omental Fat (OM), expose the omental/pancreatic complex by extending the spleen from the peritoneal cavity with forceps (Figure 1C). Release omentum from the pancreas and spleen by closely trimming all tissue connections. Ensure that any remaining pancreatic tissue is excised.</li>
	<li>For the Gonadal Fat (GF), use forceps to lift the gonadal fat surrounding the ovaries and excise by cutting tissue connections (Figure 1A upper right).</li>
	<li>For the Uterine Fat (UF), use forceps to lift the uterine fat surrounding the uterine horns and excise by cutting tissue connections (Figure 1A lower right).</li>
	<li>For the Mesenteric Fat (MF), cut the junction between the small intestine and the pylorus. Use forceps to firmly grip the free end of the small intestine, and slowly &#34;peel&#34;&#160;it away from the mesenteric fat. Release the mesenteric fat from the mesenteric root using dissecting scissors (Figure 1A lower left).</li>
	<li>For the Splenoportal Fat (SF), lift the distal end of the spleen using forceps to expose the thin fatty band of tissue connecting the hilum of the spleen to the pancreas. Excise the splenoportal fat by first releasing it from the pancreas and then carefully dissecting it from the spleen (Figure 1B).</li>
</ol>

<table><tbody><tr><td>Dissection Tools</td><td>&amp;nbsp;Fine Science Tools</td><td>NA</td><td></td></tr><tr><td>PBS</td><td>&amp;nbsp;Corning</td><td>21-040-CV</td><td>Without calcium and Magnesium</td></tr></tbody></table>

harvesting peritoneal fat depots: a technique to study ovarian cancer colonization of peritoneal adipose tissues in mouse models

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">In Vivo</a><a href="https://www.jove.com/t/52721">&#160;and&#160;Ex Vivo&#160;Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we use a euthanized female mouse model bearing ovarian cancer. We then describe a protocol to identify and extract fat depots or adipose tissues associated with the five primary peritoneal organs to study metastatic cancer cell colonization.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20378/20378_Figure1.jpg" /> 
Figure 1.&#160;The relative locations and structures of the main peritoneal adipose depots. (A)&#160;Gross anatomic dissection showing the relative location of four of the five primary sources of peritoneal fat. The omentum (OM; outlined), gonadal fat (GF), uterine fat (UF), mesentery (MY), ovary (ov), uterine horns (uh), and small intestines (si) are shown.&#160;(B)</stro...

Harvesting Peritoneal Fat Depots: A Technique to Study Ovarian Cancer Colonization of Peritoneal Adipose Tissues in Mouse Models

Source: Krishnan, V. et al. In Vivo&#160;and&#160;Ex Vivo&#160;Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in ...

The adipose tissue or the fat depots primarily present in the omental, mesenteric, uterine, gonadal, and splenoportal regions of the peritoneum may provide a suitable microenvironment for cancer cell colonization during metastasis.
To isolate these peritoneal fat depots, begin by taking a euthanized female mouse. Make a midline surgical incision through the peritoneal wall to expose its internal organs. Now, locate the omental fat present over the stomach and pancreatic spleen complex and extract it. Excise the gonadal fat surrounding the ovaries. Further, remove the uterine fat enveloping the uterine horns. Subsequently, cut the junction between the small intestine and pylorus to release the mesenteric fat attached to the intestine. Finally, extract the thin band of splenoportal fat connecting the distal end of the spleen with the pancreas.
Maintain the dissected fat tissues in a suitable chilled buffer to preserve tissue physiology. Analyze these peritoneal fat tissues to assess the presence of colonized metastatic cancer cells.

harvesting-peritoneal-fat-depots-technique-to-study-ovarian-cancer

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Animal model studies

1.2K 조회수. 출처: Krishnan, V. et al. In Vivo 및 복막 지방에서 은하수 반점 구조의 난소암 전이성 집락을 연구하기 위한 생체 외 접근 방식. J. Vis. 특급 (2015) 이 비디오에서는 난소암을 앓고 있는 안락사된 암컷 마우스 모델을 사용합니다. 그런 다음 전이성 암세포 집락화를 연구하기 위해 5개의 주요 복막 기관과 관련된 지방 저장소 또는 지방 조직을 식별하고 추출하는 프로토콜을 설명합니다...

비디오: 복막 지방 저장소 채취: 마우스 모델에서 복막 지방 조직의 난소암 집락화를 연구하는 기술 - 개념

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

마우스 지방 조직 수집 및 RNA 분석에 대 한 처리

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

연구

JoVE Journal

생화학

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

인간 지방 Microphysiologic 플랫폼: 백색 지방 조직에 끼여

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

행동분석학

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

비 투 열 방법을 통해 복 부 피하 지방 조직 생 검에 대 한 기술

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Harvesting Peritoneal Fat Depots: A Technique to Study Ovarian Cancer Colonization of Peritoneal Adipose Tissues in Mouse Models

내레이션 대본

Harvesting Peritoneal Fat Depots: A Technique to Study Ovarian Cancer Colonization of Peritoneal Adipose Tissues in Mouse Models