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. 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-coagulant) blood collection tubes into a 50 ml sterile conica...

periodic acid-schiff staining of cells: an in vitro technique to detect glycogen levels in peripheral blood mononuclear cells

Source: Tabatabaei Shafiei, M., et al. <a href="https://www.jove.com/v/52199">Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining.</a> J. Vis. Exp. (2014).
This video demonstrates the periodic acid-Schiff staining technique for assessing glycogen levels of peripheral blood mononuclear cells in vitro. The stain imparts a magenta color to intracellular glycogen granules, rendering them visible under a microscope.

<img alt="Figure 1" src="/files/ftp_upload/21039/21039_Figure1.jpg" /> 
Figure 1: Step by step methodology of PAS staining on PBMC.&#160;(A)&#160;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.&#160;(B)&#160;Isolated PBMCs are fixed onto the slide using formalin-ethanol...

Periodic Acid-Schiff Staining of Cells: An In Vitro Technique to Detect Glycogen Levels in Peripheral Blood Mononuclear Cells

Source: Tabatabaei Shafiei, M., et al. Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining. J. Vis. Exp. (2014).
...

Peripheral blood mononuclear cells, or PBMCs, are circulatory blood cells with single, large nuclei, comprising B and T lymphocytes, natural killer cells, monocytes, and dendritic cells. PBMCs store surplus glucose in polymeric form as glycogen - a branched polysaccharide - in their cytoplasm.
To detect glycogen presence in PBMCs, prepare a uniform smear of isolated PBMCs on a glass slide. Treat the cells with formalin - a fixative solution, that cross-links cellular biomolecules, preserving PBMCs&#8217; structural integrity.
Partially immerse the slide in amylase solution. This enzyme enters cells and hydrolyzes glycogen in the treated cells. This results in two distinct cell populations - enzyme-treated, glycogen-deficient, and non-amylase-treated, glycogen-rich cells.
Overlay them with periodic acid solution which oxidatively converts the hydroxyl groups of glycogen molecules to aldehydes.
Rinse the slide in water to halt the oxidation reaction and remove excess periodic acid. Treat the slide with Schiff reagent, which reacts with the aldehyde groups of periodic acid-treated glycogen, generating an insoluble magenta-colored complex.
Apply mounting media to restrain cells during imaging. Place a coverslip to spread the mounting media over the entire slide surface for better visualization. Image the stained cells.
Non-amylase-treated cells contain magenta glycogen granules, absent in enzyme-treated cells.

periodic-acid-schiff-staining-cells-an-vitro-technique-to-detect

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Staining Techniques

Cell staining

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

JoVE Journal

Biochemistry

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Medicine

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

Periodic Acid-Schiff Staining of Cells: An In Vitro Technique to Detect Glycogen Levels in Peripheral Blood Mononuclear Cells

Transcript