One Step Polymer HRP Mouse and Rat IgG (H+L)

Procedure: IHC/ICC procedure for frozen, paraffin sections and cell smears. 1. Deparafinize and hydrate tissue sections through xylene or other clearing agents and graded alcohols.(For frozen sections or cell smears; use unfixed, acetone fixed or appropriate fixative for the antigen in question; for cell smears it may be necessary to permealize the cell by detergent, please refer to antibody protocol). 2. Wash 2-3 with distilled or deionized water. 3. Incubate sections/cell smear with Endoblocker (#1) for 5-10 minutes at room temperature (RT). Wash with distilled water 3X. 4. Note: If antigen retriever (Trypsin AR-6541, Pronase AR-6542, Pepsin AR-6543, Citrate buffer AR-6544, Buffer w EDTA pH 8.5 AR-6545, Tris buffer pH 10 AR-6546) is required it can be applied at this step. Please refer to data sheet for the primary antibody. 5. Wash slide with PBS or Tris saline buffer (with 0.02-0.05% nonionic detergent, Triton X100, Tween 20 or NP-40) or washing buffer (Immuno Automation buffer IBSC cat # AR-6561) 3X. 6. Incubate sections/ cell smear in Protein blocking solution (#2), for 5-10 minutes at RT. 7. Incubate sections/cell smear with primary antibody (NOT SUPPLIED, ONLY BUFFER IS SUPPLIED FOR DILUTION) for 20-30 minutes at RT. (For more information, refer to instructions for primary antibody).The primary antibody dilution buffer supplied can also be used as a negative control. 8. Wash slide with PBS 5-7X 9. Incubate with One-Step HRP polymer (#4) for 20-30 minutes at RT. 10. Wash slide 5-7 times with buffer. Caution: Peroxidase reagents are destroyed by sodium azide and should be avoided in all buffers and regents. 11. Wash slide with deionized or distilled for 2-3X. 12. Incubate with AEC or DAB chromogen reagent (Not supplied). 13. Wash 5-7X with buffer. 14. Incubate with counterstain (Not supplied). 15. Wash slide with tap water distilled water. 16. Mount slide with appropriate mounting medium.

Reagent: 15 mL = 150 tests when 0.1 mL is applied per slide 1. Ready-to-use, Peroxidase Block, Hydrogen Peroxide, 15 mL (white color cap) 2. Ready-to-use Protein Blocking Solution, 15 mL (blue color cap) 3. Primary Antibody Dilution Buffer green color, 25 mL (for dilution of primary antibody). Please refer to the data sheet of primary antibody for dilution. Use this buffer as a Negative Control 4. IBSC-One-step HRP-anti-Mouse and Rat Polymer, 15 mL (orange color cap) Reagents required but not supplied: Washing buffer, antigen retrievers, positive or negative control, primary antibody, chromogen, counterstain and mounting medium

Immunohistochemistry (IHC)/Immunocytochemistry (ICC) is the localization of antigens by the use of antigens in tissue sections/cells by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold. Several IHC techniques are commonly used: labeled biotin secondary antibody streptavidin-peroxidase (LBSASP), HRP anti-HRP, ABC, catalyzed signal amplification, polymer system and others, to detect antigens on tissue and cell. In this kit the first layer is unlabeled primary antibody, the second layer is biotinylated secondary antibody, the third layer is Enzyme-Streptavidin conjugate (HRP-Streptavidin) to replace the complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solution to produce different colorimetric end products.