eoe sub articles

EoE Sub Articles

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Gross Anatomy of the Prostate and Individual Lobe Microdissection
NOTE: This is depicted in Figures 1(i-n) and Figure 2
<ol>
	<li>Flip the tissue with a pair of blunt forceps so that the dorsal side faces up, showing the dorsal lobes, which resemble a butterfly&#8217;s wings.</li>
	<li>Collect the dorsal lobes by holding the lobe with forceps and snipping at the base with scissors (Figure 1j).</li>
	<li>Flip over the remaining tissue to the ventral side.</li>
	<li>Collect the lateral lobes, which are small and usually wrap the urethra on the side, which are wedged between the anterior, ventral, and dorsal lobes (Figure 1k).</li>
	<li>Next, collect the ventral lobes, which are larger than the lateral lobes and lie on the urethra ventrally (Figure 1l).</li>
	<li>Last, harvest the anterior lobes, the largest of the four, by cutting and discarding the urethra (Figure 1m).</li>
	<li>Process the tissue pieces according to experimental needs.</li>
</ol>

<table><tbody><tr><td>Mouse surgical instruments (Mouse Dissecting kit)</td><td>World Precision Instruments</td><td>MOUSEKIT</td><td></td></tr><tr><td>Dissection microscope</td><td></td><td></td><td></td></tr><tr><td>RPMI medium</td><td>Thermofisher Scientific</td><td>11875093</td><td></td></tr><tr><td>Dissection medium (DMEM + 10%FBS)</td><td>Thermofisher Scientific</td><td>11965-084</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>10438018</td><td></td></tr><tr><td>PBS (Phosphate buffered saline)</td><td>Thermofisher Scientific</td><td>10010031</td><td></td></tr><tr><td>Collagenase</td><td>Thermofisher Scientific</td><td>17018029</td><td>Make 10x stock (10mg/ml) in RPMI, filter sterilize, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Syringes and Needles</td><td>Fisher Scientific</td><td></td><td></td></tr></tbody></table>

individual lobe extraction from prostate: a technique to harvest individual prostate lobes from mouse prostate

Source: Nath, D. et al. <a href="https://www.jove.com/video/58397" target="_blank">Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models.</a> J. Vis. Exp. (2018)
This video describes a technique to harvest individual lobes from the prostate gland of a mouse model. Individual lobes can then be used for histological analysis and cancer research.

<img alt="Figure 1" src="/files/ftp_upload/20394/20394_Figure1.jpg" /> 
Figure 1: Dissection of the mouse prostate. Step-by-step images for the dissection of the mouse prostate from the UGS: (a) removing fat, (b) removing the urinary bladder, (c) removing the vas deferens, (d) ventral view of the prostate with the urethra, (e) dorsal view of the prostate with the urethra, (f-...

Individual Lobe Extraction from Prostate: A Technique to Harvest Individual Prostate Lobes from Mouse Prostate

Source: Nath, D. et al. Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models. J. ...

In rodents, the prostate gland is located around the base of the urinary bladder and surrounds the top portion of the urethra. The gland has four distinct pairs of lobes. Based on their anatomical orientation relative to the urinary bladder, they are called the ventral, lateral, anterior, and dorsal lobes.
The ventral lobes lie anterior to the urethra and inferior to the bladder. Two lateral prostate lobes, flanking the ventral lobes, reside on both sides of the urethra. The anterior lobes are translucent tubular structures attached to the seminal vesicles. The dorsal lobes, resembling butterfly wings, are positioned on the posterior side at the base of the seminal vesicles.
To harvest individual prostate lobes, begin by taking a mouse&#39;s urogenital system in a Petri dish. Use a dissecting microscope to distinguish the different lobes. Position the dorsal side to face upward. Locate the two dorsal lobes. Use scissors to excise them at their base. Now, invert the prostate tissue to face the ventral side. Locate the pair of lateral lobes. Use scissors to snip the lobes and separate them. Next, locate the ventral lobes and excise them. Lastly, separate the anterior lobes present at the top.

individual-lobe-extraction-from-prostate-technique-to-harvest

Research

JoVE Encyclopedia of Experiments

Cancer Research

Prostate Cancer

Animal model studies

1.3K ビュー. 出典: Nath, D. et al. Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models. J. Vis. Exp. (2018) このビデオでは、マウスモデルの前立腺から個々の葉を採取するテクニックについて説明しています。その後、個々の葉を組織学的分析やがん研究に使用できます。 

ビデオ: 前立腺からの個々のローブ抽出:マウスの前立腺から個々の前立腺ローブを採取する技術 - 概念

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

癌細胞の3Dスフェロイド培養における細胞生存率と死の評価

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

JoVE Journal

がん研究

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

手術標本からヒト膵島のレーザーマイクロダイセクションのために改良されたプロトコル

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

医学

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3次元組織学ボリュームとマウス乳腺研究への応用再建

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Individual Lobe Extraction from Prostate: A Technique to Harvest Individual Prostate Lobes from Mouse Prostate

記録

Individual Lobe Extraction from Prostate: A Technique to Harvest Individual Prostate Lobes from Mouse Prostate