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Dear Sir, 1. Please describe the problem (high background, no staining etc). No staining 2. On what material are you testing the antibody in IHC? Species? Cell line? Lymphoblast (Human) Tissue? 3. How did you fix the samples? Ethanol, methanol (Tried) Acetone (Tried) Paraformaldehyde Other (10%TCA) 4. Did you apply antigen retrieval step? Enzymatic method Heat mediated technique Other 5. How did you block the unspecific binding sites? Using 10%BSA in PBS+ 0.1% Tween 20 6. Primary antibody Specification (in which species was it raised against)? Mouse At what dilution(s) have you tested this antibody? 1:100, 1:500 and 1:1000 Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? 2h. 5min washing with PBST for 15min. 7. Secondary antibody What secondary antibody are you using? anti-mouse (TR-labelled) Specification (in which species was it raised against)? goat At what dilution(s) have you tested this antibody? 1:100, 500 & 1000 Incubation, wash steps? 0.5h and 1h at R.T and 37C Do you know whether the problems you are experiencing come from the secondary? yes, since I do get signal using another aliquote of the treated cells but using another secondary Ab. What detection method are you using? Fluoresence microscopy. 8. Background staining Please provide an image of your staining I do not have a picture (As it is almost faint red with no indication about cells. However I do get very nice stainíng from DAPI. 9. Which detection system did you use? Fluoresence microscopy. 10. Did you apply positive and negative controls along with the samples? Please specify. Yes, DAPI staining to prove that I have cells on the slides. double staining the same slide with another antibody against another target and it works. 11. Optimization attempts How many times have you tried the IHC? 15 times Do you obtain the same results every time? What steps have you altered? Fixation, primary and secondary dilutions I am looking forward to hearing from you soon. Thank you for your offer. Yes I do like to try it. You can send it to the below address:
Asked on Apr 26 2004
Thank you for your e-mail and for providing more information of your protocol. I have placed an order for your -one vial of replacement vial of ab5884. For your information, the new order number is: 40004. Please do let me know how you are getting on with this replacement.
Answered on Apr 27 2004