RE: Enzyme Histochemistry for Glycogen Storage Diseases

From:"Smith, Allen"

T. Takeuchi (1958) Histochemical demonstration of  branching enzyme (amylo-1,4--1,6-transglucosidase) in animal tissues. J.Histochem.Cytochem. 6: 208-216.
Incubating medium:
    50 mg. glucose-1-phosphate (Na or K salt)
    10 mg 5'-adenylic acid (free acid)
    4 mg soluble glycogen
   15 ml distilled water
   10 ml 0.1 M pH 5,8 acetate buffer
Lugol's Iodine
    1 g iodine
    2 g potassium iodide
  100 ml water
        Tissue may be stored at -20 degrees C for up to 36 hr. but no longer.
    1. Incubate 10-40 micron fresh frozen sections in the incubating medium 1hr at room temperature.
    2. Stain 5 min. in 10% Lugol's iodine
        Sections can be mounted in glycerol gel and examined immediately or mounted permanently. [A.A. Smith, E.M. Perkins,    
        and H.Machida (1966) Durable mounts of the iodine stain for the phosphorylase reaction. Stain Technol. 41: 346-348.]
    3. 0.1% iodine in 70% ethanol, 3 min.
    4. 0.1% iodine in 95% ethanol, 5 min.
    5. 0.1% iodine in absolute ethanol, 2 changes, 5 min. each.
    6. 0.1% iodine in xylene, 2 changes, 5 min. each
    7. Mount in Clay-Adams "Histoclad" containing 1 mg iodine per ml of resin. (Hartman-Leddon's "Histoclad"
           can substituted. Neither "Permount" nor balsam works.)
Red-brown deposits indicate strong branching enzyme activity.  Brown deposits indicate moderate branching enzyme activity.
Black, gray, or blue deposits indicate little or no branching enzyme activity.

Allen A. Smith, Ph.D.
Barry University
School of Graduate Medical Sciences
    Podiatric Medicine and Surgery
Miami Shores, Florida  33161-6695

-----Original Message-----
From: Amy Porter []
Sent: Wednesday, April 23, 2003 3:08 PM
To: Histonet
Subject: Enzyme Histochemistry for Glycogen Storage

Does anyone out there in histo land know of a procedure for staining "brancher" and "debrancher" enzymes....or are these strictly metabolic tests that have to be performed on ground up flash frozen tissue?

Amy S.Porter, HT(ASCP)
Michigan State University
College of Human Medicine

Do you Yahoo!?
The New Yahoo! Search - Faster. Easier. Bingo.

<< Previous Message | Next Message >>