Question (21363) | Anti-CD45RA antibody [MRC OX-33] (ab33933)

Thank you for your recent telephone enquiry. I have obtained the following general testing method from the originator of ab33933 and ab33934. Please note that this protocol is a guideline only and may need to be optimized in your laboraotry. I hope this will be helpful to you. Should you have any further questions, please do not hesitate to contact us. 1. De-paraffinise sections thoroughly in Xylene/synthetic solvent and hydrate through graded series of alcohols. 2. Wash twice in TBS. 3. If required, treat with 0.3% (w/v) hydrogen peroxide in methanol for 30 minutes to block endogenous peroxidase activity (2% w/v H2O2/Methanol can be used for a shorter time if preferred). 4. Wash once in TBS. 5. If required, include appropriate antigen retrieval step to enhance immunostaining. 6. Wash once in TBS. 7. Incubate sections for 10 minutes in 10% normal serum from species in which secondary antibody was raised. 8. Tap excess serum off the slides before staining. 9. Incubate sections in primary antibody for at least 1 hour at room temperature in a humid chamber or overnight at 4°C. Wash three times in TBS. 10. Add enzyme conjugated secondary antibody at recommended dilution (see specific datasheet for details). Incubate for at least 30 minutes at room temperature. 11. Wash three times in TBS. 12. Incubate in appropriate substrate solution for recommended period of time (we would recommends the use of DAB substrate with HRP conjugated antibodies, and Fast Red / Napthol AS-MX for Alkaline Phosphatase conjugated antibodies). 13. Wash once in water. 14. Counterstain in haematoxylin, 2-10 minutes. ‘Blue’ in running water for 5 minutes. 15. Mount in aqueous mounting medium or alternatively dehydrate through alcohols and xylene/solvent and mount in synthetic mountant. Notes: NB. Note that certain substrates are soluble in alcohol - please refer to supplier information for details. Appropriate control samples should always be included. It may be useful to include a control in which no primary antibody is used at all, to determine any non-specific binding of the secondary reagent to the target tissue. Solutions used: TBS (stock solution ×10 concentrated) Sodium chloride 87.66 g, Tris 60.55 g, Distilled water 1 litre. Adjust pH to 7.5 using concentrated HCl.