Name: Video: Vibratome Sectioning of Whole Murine Kidney: A Technique to Generate Thin Sections of Murine Kidney - Concept
Uploaded: 2023-04-30T13:50:45.000+00:00
Duration: 1 min 48 s
Description: 1.3K Views. Source: Grainger, N. et al. Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning. J. Vis. Exp. (2021) In this video, we demonstrate the vibratome sectioning of a whole mouse kidney to obtain thin slices. These sliced tissue sections can be stored at low temperatures until further downstream analysis.

eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Prepare Solutions
<ol>
	<li>Prepare 1 L of Krebs-Ringer Bicarbonate (KRB) solution containing 120.35 mM NaCl, 5.9 mM KCl, 15.5 mM NaHCO3, 1.2 mM Na2HPO4,1.2 mM MgCl2, 11.5 mM glucose, and 2.5 mM CaCl2. On the day of use, maintain the KRB solution on ice. 
	NOTE: KRB can be stored at 4 &#176;C for up to one week and should be pre-bubbled with a mix of 97% O2&#160;and 3% CO2&#160;for at least 10 min prior to use.</li>
</ol>
2. Vibratome Sectioning of the Kidney
<ol>
	<li>Use blunt-ended forceps to gently grasp and remove the prepared kidney from the ice-cold KRB solution. Immediately place the kidney on absorbent paper for ~2&#8211;4 s to remove excess external moisture. Gently roll the kidney across the absorbent paper to ensure that all sides of the parenchyma have dried so that there is optimal adhesion of the kidney to the vibratome stage. 
	NOTE: As the renal pelvis is located inside the kidney and therefore protected by the outer parenchyma, this short drying period would not be detrimental to tissue integrity.</li>
	<li>Immediately apply a thin layer of cyanoacrylate glue (~1 cm2) to the base of the vibratome specimen plate and use blunt-ended forceps to place the kidney, ureter side down, on the area covered in glue. Gently apply downward pressure to the top of the kidney with the flat edge of the forceps for approximately 10&#8211;20 s to dry the glue. 
	NOTE: To stabilize the kidney during this procedure, use an additional pair of forceps to keep the kidney upright as the glue dries. It is critical that the kidney adheres to the specimen plate in an upright position so that sections are cut straight. To ensure that the kidney has successfully adhered to the specimen plate, gently push the side of the kidney. If the kidney has successfully adhered to the plate, the base of the kidney should stay secured to the plate.</li>
	<li>Firmly secure the specimen plate to the bottom of the buffer tray. Adjust the level of KRB solution so that the top of the kidney is fully immersed. During sectioning steps, as the vibratome blade moves deeper into the buffer tray, remove KRB solution so that the blade holder does not become immersed in solution. 
	NOTE: If the glue has not completely dried before proceeding with this step, the glue will often move up from the base and onto the kidney parenchyma. This excess glue will make sectioning more variable. If this does happen, remove the kidney from the plate and proceed with a fresh kidney preparation.</li>
	<li>For automatic vibratome sectioning, select the start and end positions of the vibratome blade-cutting cycle. Verify these positions are ~0.5&#8211;1 cm clear of the kidney to ensure that with each blade advancement, the entire kidney plane is sectioned.</li>
	<li>Prepare a multi-well plate (24- or 48-well) by filling wells with KRB solution, and place the plate on ice. 
	NOTE: When generated, individual sections should be placed into separate wells to keep track of section depth.</li>
	<li>Start the automatic cutting process. During the initial pass of the blade, ensure that the blade makes contact with the very top of the kidney. If contact is not made, adjust the starting Z-position of the blade.</li>
	<li>Using forceps, collect sections that are liberated from the kidney. Immediately transfer the sections to individual wells and note the Z-depth of the sections to gauge the approximate PKJ location within kidney sections. 
	NOTE: Depending on the cut parameters, some sections may not be cut free from the kidney block. If this occurs, carefully use fine spring scissors to cut sections from the kidney block. Users are also encouraged to actively visualize sections under a light microscope whilst free-floating in individual wells to ensure optimal cut settings and tissue location. Sections containing the PKJ will typically be derived ~ 1000&#8211;1500 &#181;m from the top of the kidney.</li>
	<li>Continue the sectioning protocol until the PKJ regions become more apparent. Refer to the representative results section for a description of the PKJ regions as sectioning proceeds.</li>
	<li>At this point, optimize the sectioning parameters to ensure that the sections are liberated from the kidney block uniformly and are intact. Additionally, make sure that the PKJ regions are continuous and unbroken because broken PKJ walls will not allow adequate imaging of cells within the wall due to collapse. If walls become broken, decrease the sectioning speed and increase the section thickness and continue to observe sections under a light microscope to fine-tune the cutting parameters.</li>
	<li>Store sections at 4&#176;C in KRB solution until experimentation begins.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well culture plate</td>
			<td>ThermoFisher Scientific</td>
			<td>142485</td>
			<td>To store kidney slices in</td>
		</tr>
		<tr>
			<td>48-well culture plate</td>
			<td>ThermoFisher Scientific</td>
			<td>152640</td>
			<td>To store kidney slices in</td>
		</tr>
		<tr>
			<td>Absorbent paper</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td>To dry the kidney before applying glue</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Amazon</td>
			<td>B001PILFVY</td>
			<td>For adhering the kidney to the specimen plate</td>
		</tr>
		<tr>
			<td>B6;129S-Gt(ROSA)26Sor/J</td>
			<td>The Jackson Laboratory</td>
			<td>13148</td>
			<td>GCaMP3 Mice</td>
		</tr>
		<tr>
			<td>B6;129S-Gt(ROSA)26Sor/J</td>
			<td>The Jackson Laboratory</td>
			<td>24105</td>
			<td>GCaMP6f Mice</td>
		</tr>
		<tr>
			<td>B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J</td>
			<td>The Jackson Laboratory</td>
			<td>19079</td>
			<td>smMHC-CRE Mice</td>
		</tr>
		<tr>
			<td>C57BL/6-Tg(Pdgfra-cre)1Clc/J</td>
			<td>The Jackson Laboratory</td>
			<td>13148</td>
			<td>PDGFRa-CRE Mice</td>
		</tr>
		<tr>
			<td>Fine-tip forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Used for fine dissection of kidney</td>
		</tr>
		<tr>
			<td>Gillette Silver Blue double-edge blades</td>
			<td>Amazon</td>
			<td>B009XHQGYO</td>
			<td>For insertion into blade holder of vibratome</td>
		</tr>
		<tr>
			<td>Student Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>91106-12</td>
			<td>For gently holding and moving the kidney</td>
		</tr>
		<tr>
			<td>Student Dumont Forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td>Used for internal dissecting forceps</td>
		</tr>
		<tr>
			<td>Vibrocheck</td>
			<td>Leica</td>
			<td>14048142075</td>
			<td>Optional component for calibrating blade movement during cutting</td>
		</tr>
		<tr>
			<td>VT1200 S Vibrating Blade Microtome</td>
			<td>Leica</td>
			<td>14912000001</td>
			<td>Configuration 1 is used in our protocol</td>
		</tr>
	</tbody>
</table>

vibratome sectioning of whole murine kidney: a technique to generate thin sections of murine kidney

Source: Grainger, N. et al. <a href="https://www.jove.com/video/62040" target="_blank">Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the vibratome sectioning of a whole mouse kidney to obtain thin slices. These sliced tissue sections can be stored at low temperatures until further downstream analysis.

Vibratome Sectioning of Whole Murine Kidney: A Technique to Generate Thin Sections of Murine Kidney

Source: Grainger, N. et al. Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning. J. Vis. Exp. (2021)
In ...

To prepare thin sections of a mouse kidney, first, obtain a freshly harvested mouse kidney. Transfer the kidney onto a filter paper and dry it to ensure optimal adhesion to the vibratome specimen holder.
Next, mount the kidney, ureter side down, using a thin layer of cyanoacrylate glue applied to the base of the vibratome specimen holder, securing the kidney in an upright position. Transfer the specimen holder onto a buffer tray.
Fill the tray with a suitable buffer to fully immerse the top surface of the kidney. An ice bath surrounds the tray to prevent tissue damage from heat buildup during slicing. Set the vibratome blade to an appropriate angle, speed, and amplitude. Adjust the thickness parameter to obtain tissue sections of desired thickness.
During operation, the blade vibrates laterally and advances, penetrating through the tissue specimen. The vibrating blade allows the sectioning with less pressure and minimizes the tissue deformation, generating uniform kidney tissue sections of interest while maintaining the tissue morphology.
Meanwhile, assemble a multi-well plate containing a suitable chilled buffer solution to mimic the physiological conditions of the tissue. Now, transfer the sliced tissue sections into the buffer. Finally, store the tissue sections at low temperature until further downstream analysis.

vibratome-sectioning-whole-murine-kidney-technique-to-generate-thin

Research

JoVE Encyclopedia of Experiments

Cancer Research

Urinary Tract Cancer

In vitro studies

1.3K Views. Source: Grainger, N. et al. Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning. J. Vis. Exp. (2021) In this video, we demonstrate the vibratome sectioning of a whole mouse kidney to obtain thin slices. These sliced tissue sections can be stored at low temperatures until further downstream analysis. 

Video: Vibratome Sectioning of Whole Murine Kidney: A Technique to Generate Thin Sections of Murine Kidney - Concept

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

JoVE Journal

Neuroscience

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

Immunology and Infection

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

Vibratome Sectioning of Whole Murine Kidney: A Technique to Generate Thin Sections of Murine Kidney

Transcript

Vibratome Sectioning of Whole Murine Kidney: A Technique to Generate Thin Sections of Murine Kidney

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics