Name: detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining
Uploaded: 2014-12-23T15:00:00
Description: Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

Research with human blood samples was approved by Concordia University Ethics Review Board, certificate number 10000618. The work on mouse muscle was approved by Concordia University Ethics Review Board, certificate number 2010BERG.
1. PBMC Isolation from Whole Blood
NOTE: Carry out this procedure in a biosafety cabinet using sterile technique and manufacturer-sterilized equipment.
<ol>
	<li>Carefully pour 10-15 ml of whole blood from the heparinized (anti-coagula...

<ol>
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The critical steps of this video article were during washing and amylase treatment of the cells. While washing the slides, the key step was using a plastic squeezable washing bottle and letting the water gently run through the sample on the slide and not aiming directly onto the samples. Even the slightest direct water pressure would cause the cells to come off the slide. Another key step was to use the same slide for &#177; amylase conditions. After the PBMCs were adhered to the slide, the slide was carefully placed int...

Periodic acid Schiff (PAS) staining is an immunohistochemical technique that is widely used in muscle research and diagnostics. It is also utilized as a diagnostic tool on blood samples. The technique works by applying periodic acid solution to the sample, which oxidizes units within the polysaccharide creating aldehyde groups that react with the colourless Schiff&#39;s reagent thereby producing a deep magenta product. The steps of this procedure are shown in Figure 1. The stain turns anything with polysaccharides magenta, including glycogen, glycoproteins, glycolipids, mucins, or other molecules with polysaccharide moieties.
PAS staining is often used to measure glycogen levels in muscle fibers. Muscles tissue sections are ideal for the technique as they firmly attach to the slide and withstand multiple washing and staining steps. Glycogen is most present in fast twitch Type II muscle fibers, which have a high demand for rapid ATP production requiring glycogen for maximum performance1,2. Glycogen is a branched polymer of glucose that can be broken into free glucose through the action of glycogen phosphorylase enzymes. In times of rest and nutritional-sufficiency, glycogen is replenished through the process of glycogenesis, while in times of nutritional insufficiency or high-energy demand; glycogen is broken down into glucose by glycogenolysis. From as early as the 1950&#39;s clinician scientists have explored PAS staining on blood samples to analyze glycogen content in various diseases3-7. For example, in Pompe&#160;disease-a bonafide glycogen storage disease- white blood cells accumulate large amounts of glycogen that differs significantly from healthy controls8.
This video-article demonstrates an adapted version of PAS staining for use on peripheral blood mononuclear cells (PBMC) samples from venous blood of healthy human subjects. PBMCs contain mostly lymphocytes of the T lymphocyte and B lymphocyte families, as well as other immune cells such as natural killer cells and monocytes. The first purification step removes erythrocytes, neutrophils, and other granulocytes. This technique provides data on a concentrated proportion of lymphocytes allowing for more robust enumeration of PAS-positive cells as compared to using whole blood smears.
<img alt="Figure 1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> 
Figure 1: Step by step methodology of PAS staining on PBMC. (A) First, isolation of PBMC is achieved through ficoll gradient, the left panel shows the preparation before centrifugation, the right panel shows it after centrifugation where the buffy coat containing the PBMC is observed in the center of the tube. (B) Isolated PBMCs are fixed onto the slide using formalin-ethanol fixative solution. The slide is gently rinsed with distilled water from a plastic wash bottle. (C) The slide is then placed in a 100 ml beaker half way filled with amylase solution, which will dissolve glycogen. The slide is gently rinsed. (D) The slide is treated with periodic acid solution, where oxidation of saccharides takes place. Slides are gently rinsed; this will remove the excess periodic acid and stop the oxidation step. (E) When the Schiff reagent is added to the slides, it will react with aldehydes created during the oxidation step. This colorless reagent will then result in a deep red magenta product. Slides are gently rinsed to remove the excess Schiff reagent.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

To validate the reagents and basic technique, PAS staining was performed according to manufacturer&#8217;s instructions on mouse soleus muscle sections. The staining was done same day as the sacrifice and in the last step of the staining, the sections were fixed by xylene. The soleus muscle is known to contain ~35% glycogen-positive cells9. The stained muscle cells displayed two distinct PAS-positive features- punctate granules within the cell, and a continuous line demarking the cell membrane (Figure ...

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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