Exalpha Biologicals, Inc.

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FIX&PERM Cell Fixation and Permeabilization Kit

Flow cytometric analyses with monoclonal antibodies were so far mainly restricted to cell surface molecules. Intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins etc. were largely excluded from such studies. Also excluded from flow cytometric studies were cytoplasmic localizations of well-established membrane molecules like CD3 and CD22, which, in their cytoplasmic form, are the most reliable lineage markers in undifferentiated leukemia. With the FIX&PERM® Kit flow cytometric analysis of intracellular antigens has become as easy as surface antigen studies. The only prerequisite is the availability of suitable antibody conjugates. Most of the available monoclonal antibody conjugates can be used with the FIX&PERM® Kit, some determinants are sensitive, however, to the fixation step involved. This and the optimal fixation time have to be tested for each reagent.

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Periodic Acid Schiff Protocol

PRINCIPLE:
This type of staining is used for the detection of glycogen. Tissue sections are first oxidized by 0.5% periodic acid solution. The oxidative process results in the formation of aldehyde groupings through carbon-to-carbon bond cleavage. Free hydroxyl groups should be present for oxidation to take place. Oxidation is completed when it reaches the aldehyde and the aldehyde groups are detected by the Schiff reagent. A colorless, unstable dialdehyde compound is formed and then transforms to the colored final product through restoration of the quinoid chromophoric grouping.

QUALITY ASSURANCE:
The PAS stain, along with diastase or amylase digestion has histochemical specificity for glycogen. Skeletal muscle normally contains glycogen and is often recommended as a positive control tissue.

SPECIMEN REQUIRED:
Snap-frozen human striated muscle.

METHOD:
Technique:

From the snap-frozen biopsy, cut 10-16 micron (12 µm) sections in a crytostat. Place one or more sections to a No.1, 22 mm square coverslip.

Equipment:
  • Ceramic staining rack - Thomas Scientific #8542-E40
  • Columbia staining dish - Thomas Scientific #8542-C12
  • Columbia staining dish (jar) - Thomas Scientific #8542-E30
  • Forceps
  • Latex gloves
Reagents:
  • Absolute alcohol (100% ethanol) - Quantum, FLAMMABLE store in flammable cabinet at room temperature
  • Glacial Acetic Acid -Fisher A507-500, CORROSIVE store at room temperature
  • Amylase - Sigma A-6505, store at room temperature
  • Chloroform - Baxter 049-4, FLAMMABLE CARCINOGEN, store in a flammable cabinet at room temperature)
  • Periodic Acid - Sigma P7875, store at room temperature
  • Permount - Fisher SP15-100, FLAMMABLE HEALTH HAZARD
  • Reagent alcohol, ACS - histological Fisher A962-4 or HPLC A995, FLAMMABLE, TOXIC, TERATOGENIC, store in flammable cabinet at room temperature
  • Schiff Reagent - Harleco 6073/71, store at room temperature
  • Xylenes - Fisher #HC700-1GAL, FLAMMABLE, store in flammable cabinet at room temperature
Solutions:
  1. Carnoy's Fixative (store at room temperature) PREPARE IN A FUME HOOD
    100% Alcohol ~ 60 ml
    Chloroform ~ 30 ml
    Glacial acetic acid ~10 ml
  2. Periodic Acid Solution, 0.5 % (w/v) PREPARE FRESH FOR EACH STAIN
    Periodic acid ~ 50 mg
    Dissolved in deionized water ~ 10 ml
  3. 50% Alcohol
    Reagent alcohol ~ 50 ml
    Deionized water ~ 50 ml
  4. 70% Alcohol
    Reagent alcohol ~ 70 ml
    Deionized water ~ 30 ml
  5. 80% Alcohol
    Reagent alcohol ~ 80 ml
    Deionized water ~ 20 ml
  6. 95% Alcohol
    Reagent alcohol ~ 95 ml
    Deionized water ~ 5 ml
Staining Procedure
  1. Place the coverslip with the section on it into a columbia staining dish (Thomas Scientific (#8542- E40).
  2. Add enough Carnoy's fixative to cover the coverslip and incubate for 10 minutes. (Specific Volume? Enough to cover section?)
  3. Very carefully, rinse the (coverslip, dish, section?) with several exchanges of deionized water. Sections may wash off!!
  4. Add enough Periodic Acid solution to cover the coverslip and incubate for 10 minutes.
  5. Very carefully, rinse the coverslip with several exchanges of deionized water. Sections may wash off!! Add enough Shiff Reagent to cover the coverslip and incubate for 5 minutes
  6. Carefully wash with three exchanges of tap or deionized H2O.
  7. Dehydrate coverslip in ascending alcohol solutions (50%, 70%, 80%, 95% x 2, 100% x 2) in columbia staining dish(jar)es - Thomas Scientific #8542-E30.
  8. Rinse with xylene (3 - 4 x ) also in columbia staining dish(jar) - Thomas Scientific #8542-E30.
  9. Mount coverslip onto a labeled glass slide with Permount or any suitable organic mounting medium

Results:
Glycogen, neutral mucosubstances, basement membranes, collagen fibers, glycolipids and phospholipids will be demonstrated through a range of pink, red or purple colors. If diastase or amylase is used as a negative control, the glycogen deposits are removed leaving only the plasma membrane, which will stain pink. The two major types of fibers are usually distinguished by different intensities of staining.

REFERENCES:
  1. Thompson, Samuel W. SELECTED HISTOCHEMICAL AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield, IL, 1966
  2. Sheehan, D.C. and Hrapchak, B.B., THEORY AND PRACTICE OF HISTOTECHNOLOGY, 2nd Edition; Battelle Memorial Institute, Columbus, OH, 1987