Name: 视频: 肾脏平装制备:解剖和准备用于成年斑马鱼肾脏显微镜分析的平装程序 - 概念
Uploaded: 2023-04-30T13:53:52.000+00:00
Duration: 1 min 35 s
Description: 961 次观看. 来源:McCampbell， K. K. et al.成年斑马鱼肾脏肾单位组成和功能分析。J. Vis. Exp. （2014 年） 该视频演示了成年斑马鱼肾脏平面支架的解剖和制备。准备好的平面支架可以在显微镜下观察以进行样品分析。

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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. Dissection and Flat Mount Preparation of the Adult Zebrafish Kidney from an Unfixed Animal Sample to Visualize Fluorescent Dextran Labeling of the Proximal Tubule
<ol>
	<li>Euthanize the dextran-injected adult zebrafish by placing it into a dish with 0.2% Tricaine pH 7.0 for 4&#8211;5 min. 
	NOTE: Ensure that the fish has been euthanized before proceeding, by monitoring carefully whether the gills have ceased movement and the heart has stopped beating.</li>
	<li>Using a plastic spoon, lift the fish from the Tricaine solution, decant the solution and place the animal on a tissue or paper towel.</li>
	<li>Use a sharp pair of dissection scissors to make a cut behind the gill operculum and remove the head.</li>
	<li>Immediately open the body of the fish with the dissection scissors by making a long ventral incision from the head to the base of the caudal fin.</li>
	<li>Remove the internal organs of the fish using a pair of fine forceps.</li>
	<li>Use super-fine dissection needles to pin open the body walls to allow visualization of the kidney organ, which is adherent to the dorsal wall of the animal.</li>
	<li>Use fine forceps to detach the kidney from the dorsal wall.</li>
	<li>Gently place the kidney into a 5 mL glass vial containing 1X PBS and wash 3 times with 3&#8211;5 mL of fresh 1X PBS for 5 min each. 
	NOTE: The use of a glass vial for handling of adult kidney samples facilitates visualization of the tissue and allows for washes with large volumes (relative to the tissue mass) of approximately 3&#8211;5 mL. Alternatively, a 12-well cell culture dish can be used. While a plastic microcentrifuge tube could be used, the standard sized tubes (1.5 mL) could restrict the washing.</li>
	<li>Remove the kidney with a transfer pipette and place onto a clean glass slide with 1&#8211;2 drops of 1X PBS.</li>
	<li>Use fine forceps, flattened the kidney on the slide, making sure no tissue has curled or otherwise overturned on itself. 
	NOTE: Alternatively, a tungsten wire tool can be used to make small incisions in the connective tissue to facilitate flattened positioning of the kidney.</li>
	<li>Place small pieces of modeling clay on each corner of an 18 x 18 mm glass coverslip and slowly set the coverslip onto the kidney. 
	NOTE: Slightly angle the coverslip during placement to minimize trapping air bubbles in the solution. The modeling clay divots are approximately 2 mm in diameter (Figure 1A). Alternatively, divots of vacuum grease can be substituted in lieu of modeling clay. Add an additional drop of 1X PBS to fill the space between the coverslip and glass slide if necessary.</li>
	<li>Observe and/or image the kidney by placing the glass slide into the field of view on a stereomicroscope or compound microscope with the appropriate filter.</li>
</ol>

<table><tbody><tr><td>1X PBS</td><td></td><td></td><td>Made by diluting 10 X PBS in distilled water.</td></tr><tr><td>Fine Forceps&amp;nbsp;</td><td>Roboz&amp;nbsp;</td><td>RS-1050</td><td>Dumont Tweezers Pattern #55</td></tr><tr><td>Glass slide&amp;nbsp;&amp;nbsp;</td><td>Thermo-Fisher</td><td>4445</td><td>White Frost</td></tr><tr><td>Glass coverslip&amp;nbsp;</td><td>Thermo-Fisher&amp;nbsp;&amp;nbsp;</td><td>12-540A</td><td>18 x 18 mm</td></tr><tr><td>Modeling clay&amp;nbsp;&amp;nbsp;</td><td>Hasbro</td><td>Playdoh</td><td>Other modeling clays can be substituted and work similarly.&amp;nbsp;</td></tr><tr><td>Slide holder&amp;nbsp;&amp;nbsp;</td><td>Thermo-Fisher</td><td>12-587&amp;nbsp;</td><td>Optional: cardboard tray to store slides flat.</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ645; SMZ1000; 83455 P-Blue GFP/DAPI; 83457 P-Endow GFP/ FITC; 83457 P-TRITC</td><td>Filter set used was as follows: Hoechst/DAPI filter was used for DAPI, propidium iodide, dextran-cascade blue and alkaline phosphatase detection. GFP/FITC filter was used for dextran-FITC, dextran lucifer yellow, and antiGFP detection. The TRITC filter was used for dextran-fluoro-ruby, PI, and DBA detection.</td></tr><tr><td>Compound microscope&amp;nbsp;</td><td>Nikon</td><td>96310 C-FL UV-2E/C DAPI; 96311 C-FL B-2E/C FITC; 96313 C-FL Y-2E/C Texas Red</td><td>Filter set used was as follows: Hoechst/DAPI filter was used for DAPI, propidium iodide, dextran-cascade blue and alkaline phosphatase detection. GFP/FITC filter was used for dextran-FITC, dextran lucifer yellow, and antiGFP detection. The Texas Red filter was used for dextran-fluoro-ruby, PI, and DBA detection.</td></tr></tbody></table>

kidney flat mount preparation: a procedure to dissect and prepare a flat mount for microscopic analysis of adult zebrafish kidney

Source: McCampbell, K. K. et al. <a href="https://www.jove.com/video/51644" target="_blank">Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney.</a>&#160;J. Vis. Exp. (2014)
This video demonstrates the dissection and preparation of a flat mount of the adult zebrafish kidney. The prepared flat mount can be visualized under the microscope for specimen analysis.

<img alt="Figure 1" src="/files/ftp_upload/20573/20573_Figure1.jpg" /> 
Figure 1.&#160;Adult zebrafish kidney flat mount preparation and application to visualize conjugated dextran uptake in the PCT segment of kidney nephrons. (A)&#160;Brightfield image of a kidney specimen flat mount preparation, in which the organ has been positioned flat on a glass slide, with a coverslip placed on top of the tissue that is resting on four divots of modeling clay (hot pink color)....

Kidney Flat Mount Preparation: A Procedure to Dissect and Prepare a Flat Mount for Microscopic Analysis of Adult Zebrafish Kidney

Source: McCampbell, K. K. et al. Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney.&#160;J. Vis. Exp. (2014)
This video ...

The adult zebrafish contains a single, flattened kidney attached to the dorsal body wall through connective tissues. It consists of three main regions: head, trunk, and tail. The functional unit of the kidney, nephrons, are highly branched and drain into two major collecting ducts.&#160;
To isolate the kidney, first, prep a euthanized adult zebrafish with its ventral side facing up. Make an incision behind the operculum - a bony flap covering the gills - to remove the head. Subsequently, make another incision from the head to the base of the caudal or tail fin to expose and remove the internal organs.
Now, pin open the body walls to visualize the kidney. Dissect the underlying connective tissue to detach the kidney from the dorsal wall. Transfer the organ into a vial containing a suitable buffer, washing it thoroughly to remove debris. The buffer helps provide physiological conditions. Post wash, place the kidney, hydrated with buffer, on a glass slide.
Ensure that the kidney is completely flattened, making cuts in the connective tissue if necessary. This step aids in easy visualization during imaging. Finally, secure the organ preparation using a coverslip affixed with modeling clay pieces at its corners. The clay helps prevent tissue deformation. The flat-mounted tissue is now ready for microscopic analysis.

kidney-flat-mount-preparation-procedure-to-dissect-prepare-flat-mount

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961 次观看. 来源:McCampbell， K. K. et al.成年斑马鱼肾脏肾单位组成和功能分析。J. Vis. Exp. （2014 年） 该视频演示了成年斑马鱼肾脏平面支架的解剖和制备。准备好的平面支架可以在显微镜下观察以进行样品分析。 

视频: 肾脏平装制备:解剖和准备用于成年斑马鱼肾脏显微镜分析的平装程序 - 概念

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

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发育生物学

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

原位 斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

成人斑马鱼中西斯铂诱导的急性肾损伤模型

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Kidney Flat Mount Preparation: A Procedure to Dissect and Prepare a Flat Mount for Microscopic Analysis of Adult Zebrafish Kidney

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Kidney Flat Mount Preparation: A Procedure to Dissect and Prepare a Flat Mount for Microscopic Analysis of Adult Zebrafish Kidney