One step Polymer HRP Rabbit IgG with DAB

Preparation of DAB Chromogen Reagent 5: To one ml of buffer substrate (5BS) in a test tube, add two drop (50 µl) of reagent 5C (chromogen) mix well; this is ready-to-use DAB chromogen system. This reagent is good for 7-8 hours. 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. Rinse 2-3X with distilled or deionized water. 3. Incubate paraffin sections with Endoblocker (#1) (1-3 drops to cover section) for 10 minutes at room temperature (RT). For frozen sections use Endoblocker #1 (1:10 diluted in methanol) Rinse slide 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 10 minutes at RT. Do not Rinse the slide. 7. Incubate sections/cell smear with primary antibody (NOT SUPPLIED, ONLY Primary antibody dilution BUFFER IS SUPPLIED FOR the DILUTION) as recommended by the supplier. (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/buffer 5-7X 9. Incubate with One-Step HRP polymer (#4) for 15 minutes at RT. 10. Wash slide 5-7 times with PBS/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 DAB chromogen reagent #5 for 5-10 minutes at RT; monitor the color development under microscope. 13. Wash slides 5-7X with distilled water. 14. Incubate with appropriate counterstain (Not supplied). 15. Wash slide with tap water, distilled water. 16. Mount slide with organic or aqueous mounting medium (not supplied). (IBSC aqueous mounting medium, ImmunoHistoMount (AR-6503); Organic Mounting medium, Organo Mount (AR-6504). (Please see instructions for mounting medium)