49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands

Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490,

support@biogenex.com

SPECIAL STAINS
Toluidine Blue Stain
Cat. No. SS057
Doc. No. 932-SS057 Rev. No. A
Release Date: 24-Jun-2016

Intended Use
This product is intended for in vitro diagnostic use on the i6000  BioGenex Automated Staining System & also for Manual
staining.

Summary and Explanation

Mast cells are normally present in small numbers in the connective tissue of all organs, but particularly in the dermal layer of
skin and are identified by their cytoplasmic granules. Increased numbers of mast cells are found in many pathological conditions.
Mast cell hyperplasia in the skin (mastocytosis) manifests with skin lesions and may present with symptoms of urticaria and
flushing due to the chemical mediators released during mast cell degranulation.

Principles of the Procedure

Mast cells are not readily identified in haematoxylin and eosin stained sections as the granules are refractile and do not stain but
are but are well demonstrated by a number of special staining methods. The most common are metachromatic dye techniques
and the demonstration of chloroacetate esterase activity. Metachromatic dye such as Toluidine blue, demonstrate the strongly
sulphated acid mucopolysaccharide (heparin) content of mast cell granules. Metachromasia, tissue elements staining a different
color from the dye solution, is due to pH, dye concentration and temperature of the basic dye. Blue or violet dyes show a red
color shift, and red dyes show a yellow color shift with metachromatic tissue elements.

Reagents Provided

Potassium permanganate (HX988-YAK) 1 x 20 mL, ready to use

Potassium metabisulphite (HX989-YAK) 1 x 20 mL, ready to use
Scotts solution (HX725-YAK) 1 x 20 mL, ready to use
Toluidine blue Solution (HX990- YAK) 1 x 20 mL, ready to use

Reagents Required but Not Supplied

EZ-DeWax™ Solution (HK584-5K) 1 x 500 ml, concentrate
Super SensitiveTM Wash Buffer, 20X (HK583-5K) 1 x 500 ml, concentrate
Mounting Medium
Positive Control Tissue

Storage and Handling

Store all reagents at 2-8°C. Do not use after expiration dates as indicated on the reagent labels

Specimen Preparation
Fixation plays an important role in preserving the tissue structures to be visualized using this stain. 10% neutral buffered
formalin is the preferred fixative, however, other fixatives like Carnoy’s may also be used. Ensure that the fixed sections are
adequately embedded in paraffin. Cut tissue sections to 4-5 microns.

1
 49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands
Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490,
support@biogenex.com

Precautions
Not regarded as a health or environmental hazard under current legislation. This product contains no hazardous material at a
reportable concentration according to U.S.29 CFR 1910.1200 and EC Directive 91/155/EC.

BioGenex i6000 Automated Staining System Protocol

1. Bring reagents to room temperature. Deparaffinize the tissues and hydrate to distilled water.
2. Load the barcode-labeled slides for the appropriate stain into the BioGenex Automated Staining System slide racks. Load
the barcode-labeled reagent vials into the BioGenex Automated Staining System reagent racks.
3. Start Special Stains software on the instrument and check the protocol parameters against the factory default settings listed
below. Change any of the parameters as required. Please note that any parameter change, once saved, becomes the default
setting until changed again. Use of parameter settings other than factory default requires validation by the user.
4. Select “Start Scan” and the instrument will perform the steps listed in the table (if the factory default settings have been
selected).

Giemsa Trichrome Staining Protocol Default Setting:

Reagent No. of Incubations Incubation Time No. of After Rinses
Special Stains Wash Solution 4 30 sec. 0
Potassium permanganate 1 2 min. 3
Potassium metabisulphite 1 1 min. 3
Scotts Solution 1 5 min 2
Toluidine blue Solution 1 5 min. 2

Mounting Instructions
After staining, dehydrate and clear in xylene as usual and apply permanent mounting medium.

Manual Protocol for Alcian Blue PAS Stain

1. Deparaffinise and hydrate the slides to distilled water.

2. Apply 250µl Potassium permanganate solution & incubate for 2 minutes.
3. Rinse in distilled water
4. Apply 250µl Potassium metabisulphite solution & incubate for 1 minute. Rinse in distilled water.
5. Add 250µl Scotts solution and let stand for 5 minutes. Rinse in distilled water.
6. Add 250µl Toluidine blue solution and let stand for 5 minutes. Rinse in distilled water.
7. After staining, dehydrate and clear in xylene as usual and apply permanent mounting medium

Quality Control
A positive control slide—one that will display positive staining with this stain—should be included in every run. This
product has been validated through extensive testing on the following tissue : tissue known to contain mast cells
(neurofibroma, skin).

Trouble shooting
1. Follow data sheet instructions correctly.
2. Gently invert all reagents prior to use.

Expected Results
Toluidine blue stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining).

2
 49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands
Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490,
support@biogenex.com

Limitations of the procedure

1. The thickness of the section may affect the intensity of the staining. Necrotic tissue may exhibit non-specific staining.
2. If the positive control slide does not stain, check to make sure that the positive control slide has the correct barcode label,
applied.
3. Use tissue, which shows positive structure or element for which it is being tested.
4. All tissues need to be dewaxed. Paraffin is standard but additional time for dewaxing may be needed.

Performance Characteristics
Specificity of Special Stain
The Toluidine blue staining kit is specific and useful for identification for mast cells in skin tissue.
BioGenex has conducted studies to evaluate the performance of all its Special Stains products. BioGenex Special Stains have
shown reproducible and consistent results when used within a single run, between runs and between lots. The products have
been determined to be stable for the periods specified on the labels either by standard real time or accelerated testing methods.
BioGenex ensures product quality through 100% quality control for all products released and through surveillance programs.

Bibliography
1. Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd Ed, 1980, pp282,Battelle Press, Ohio
2. Luna L, Manual Of Histologic Staining Methods from the AFIP, 3rd Ed, 1968,pp 162-163, McGraw-Hill, NY
3. Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech