IHC Select HRP/DAB, 150 Test

Summary and Explanation:

Immunohistochemical techniques have been in use as adjunctive methods for the recognition of cells in tissue sections for at least 50 years. The first method reported use of fluorescent labels in 19421. In 1974, reports of using enzymatic labels, such as horseradish peroxidase (HRP) in routine H & E paraffin sections appeared2,3. The methods have since become the ′standard of care′ in surgical pathology when classic methods alone fail to yield a definitive diagnosis4-9.

Early techniques were based on the peroxidase-anti-peroxidase (PAP) reaction28, while improvements exploited the strong affinity of avidin for biotin10. This technique uses HRP-labeled streptavidin. The secondary antibody is conjugated with biotin and the streptavidin-HRP complex reacts with the biotin on the secondary antibodies. The resulting biotin-avidin-HRP complex can react with the primary antibody bound to the specific epitope of the target antigen. The HRP enzymes of the streptavidin complex then catalyze the substrate/chromogen reaction to form a colored reaction product (brown to black when using DAB as the chromogen) at the antigen site. A biological stain is then used to visualize the whole tissue section.

IHC Select HRP/DAB Tests for Immunocytochemistry, Immunohistochemistry & Immunohistochemistry (Paraffin).

The IHC Select Immunoperoxidase Secondary Detection System is intended for use with the CHEMICON IHC Select Prediluted Primary Antibody reagents which contain rabbit and mouse IgG for the qualitative identification of antigens by light microscopy in paraffin-embedded tissues. The IHC Select Immunoperoxidase Secondary Detection System may also be used with other rabbit or mouse IgG primary antibodies.

General Purpose Reagents

150 tests

*20783/15 mL - Blocking Reagent: normal goat serum in phosphate buffered saline (PBS) containing carrier protein

*20782/15 mL - Secondary Antibodies: biotinylated goat anti-mouse IgG and goat anti-rabbit IgG in PBS, containing carrier protein

*20777/15 mL - Streptavidin HRP: Diluted in Tris Buffered Saline

*71895/2 mL - DAB Chromogen A: 3,3′ Diaminobenzidine diluted in TBS

*71896/2 x 15 mL - DAB Chromogen B: hydrogen peroxide diluted in TBS, containing Tween^® 20

*20780/15 mL - Hematoxylin: Mayer′s hematoxylin counter stain

*20776/275 mL - 20X Rinse Buffer: TBS

Replaces: DAB225

Stability and Storage:

When stored at 2 - 8°C, the IHC Select Immunoperoxidase Secondary Detection System is stable up to the expiration date printed on the kit label. Do not freeze or expose to elevated temperatures. Discard any remaining reagents after the expiration date. Do not store kit components or perform staining in strong light, such as direct sunlight.

Precautions:

• Pooling, substitution or alteration of any reagent may cause erroneous results.

• Do not mix reagents from different lots.

• Do not allow the slides to dry at any time during the staining procedure.

• Handle all specimens and materials coming in contact with them as potentially infectious materials. Disinfect with 0.05% sodium hypochlorite.

• Do not mouth pipette reagents. If ingested, seek professional assistance or contact a Poison Control Center immediately.

• Diaminobenzidine (DAB) may be carcinogenic. This solution may cause irritation upon skin contact. Avoid inhalation, ingestion, or skin contact and wear gloves when handling DAB. If skin contact occurs, flush affected area with water. Spills may be ′neutralized′ with bleach25,26. Dispose of according to local regulations.

• Inhalation or ingestion of xylene or the highly allergenic fixative formaldehyde is harmful. These materials should be handled with gloves. Prepare in a fume hood. If skin or eye contact occurs, wash thoroughly with copious amounts of water.

• Wear disposable gloves when handling any human material and while performing the staining procedure. Materials Required:
• Primary rabbit or mouse IgG antibodies (such as CHEMICON IHC SelectPrediluted Primary Antibodies)
• Positive and negative tissue control slides
• Positively charged microscope slides
• Glass cover slips (24 x 40mm, No. 1)
• Distilled or deionized water
• Tween 20
• Permanent mounting medium, Eukitt American Histology or equivalent
• 3% laboratory grade hydrogen peroxide
• 10.0% neutral buffered formalin15
• Xylene, ACS grade
• Ethanol, absolute (100%)
• Latex, rubber or 100% nitrile gloves (xylene resistant)
• Timer
• Fume hood
• Light microscope equipped with Plan Achromat or equivalent lenses
• 60°C drying oven

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Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.