Assay & Detection

(HRP) Broad Spectrum Kit

NEW
(HRP) Broad Spectrum Kit

Article No

KDB-10019

Application

IHC

Size

1 Kit (60ml / 600 Tests)

Suggested protocols

Specifications

Application IHC
Article No KDB-10019
BioSite Brand BioSite Histo
Conjugation HRP
Description (HRP) Broad Spectrum Kit
Supplier BioSite Histo
Notes High sensitivity broad spectrum streptavidin biotin detection kitsfor primary antibodies from mouse, rabbit, rat and guniea pig. 8 ml Kits including chromogen. Ready-to-use blocking solution, sec. antibody, enzym conjugate.
Product Type Assay & Detection
Protocol Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (“Peroxide Block”). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (“Blocking Solution”). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (“Streptavidin-HRP Conjugate“). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit KDB-10015) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit KDB-10016) forms a dark brown precipitate. Staining procedure 1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
References Elias JM “Immunohistopathology – A practical Approach to Diagnosis” ASCP Press 2003 Nadji M and Morales AR Ann N.Y. Acad Sci 420:134-9, 1983 Omata M et al. Am J Clin Pathol 73(5): 626-32, 1980
Size 1 Kit (60ml / 600 Tests)
Storage 2 - 8°C
Technical Specifications High sensitivity broad spectrum streptavidin biotin detection kits for primary antibodies from mouse, rabbit, rat, and guinea pig. 8 ml kits including chromogen. Ready-to-use blocking solution, sec. antibody, enzyme conjugate.
Product Page Updated 2019-04-10T07:46:12.355Z

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