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.