Question (14913) | Anti-Desmin antibody (ab8592)

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Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific staining. When we use a rabbit Ig G as negative control we are still seeing a strong signal similar to the "postive" sample. Please see attached slides. SAMPLE Cultured myocytes from dog and cat PRIMARY ANTIBODY Rabbit polyclonal to desmin - abcam (ab8592). Initial experiments diluted antibody in 1% BSA/PBS but latterly have diluted in 5% chick serum/PBS. Have titrated primary antibody through a range of 1:25 down to 1:200. Incubation time ranged from 1h at room temp to overnight at 4deg C. Washes initially consisted of PBS (3 x 5 minute washes)but latterly have lengthened the time of the washes and have included 0.05% Tween (which significantly improves the background staining)! DETECTION METHOD Fluorescent label Alexa Fluor 500 POSITIVE AND NEGATIVE CONTROLS USED No Positives tried (myocytes should express this protein) Negatives: PBS in place of primary or Rabbit IgG in place of primary ANTIBODY STORAGE CONDITIONS +4deg C FIXATION OF SAMPLE Add enough cold methanol to cover the cell layer. Incubate at -20?C for 10 minutes. Aspirate solution. Cover cell layer with cold acetone and incubate at -20?C for 1 minute. Aspirate solution. ANTIGEN RETRIEVAL None.We're looking at cultured cells. PERMEABILIZATION STEP None BLOCKING CONDITIONS We have tried a range of BSA concentrations and by increasing the blocking time. At the lat attaempt, we tried %5 chick serum. SECONDARY ANTIBODY Molecular probes.Goat anti-rabbit IgG antibody (Alexa Fluor 500)diluted in either 1%BSA/PBS and now latterly 5% chick serum/PBS.Rnage of secodary from 1:50 down to 1:5000. Secondary incubated for 1 hour at room temperature.Washes initially were for 3 x 5 min washes in PBS but now we wash for longer using 0.05% Tween in PBS. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 7 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? The only differneces with the changes in the protocol is to reduce background fluorescence. We now are able to achieve a completely black background, but the neagative IgG fluoresces as musch as the positive antibody to desmin.

Answer

Thank you for contacting us for technical support about ab8592 and taking the time to give your protocol details and your image. I have asked for the opinion of several colleagues and we all think that the staining you have with the antibody is actually correct and that you are experiencing staining with the isotype control because of the isotype control rather than the primary antibody or because of the blocking agents. May I please suggest you run the following control to check if indeed the problem is due to the isotype control or blocking agents: run in parallel - cells incubated with 10% normal goat serum 30min then with primary antibody and then with secondary antibody -cells incubated with 10% normal goat serum 30min, then with PBS only, then with secondary antibody. You may also want to change the detergent from Tween to TritonX100 (0.3%) in the dilution buffers and try to increase the specific signal by reducing fixation to 5 min, trying to fix with 4% paraformaldehyde 10min. In our experience the Molecular probes secondary antibodies can be diluted to 1:5000 and more with excellent results. I hope the above suggestions will help, please do not hesitate to contact me again if you still experience problems with those changes.

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