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I have finally managed to find some time to fill in and fax you the questions you have asked me about the 2 antibodies that did not work, it is going through as I am writing this e mail, ab93434 and ab49873, I have also included my immuneflourescence protocol for cryosectioned slides,
I would be most grateful for your final response so I can wrap things up,
Could you please provide me with a new ab60709 to see if this antibody is still working and a trial of L glutaminase https://www.abcam.com/GLS2-antibody-ab91073.html as discussed some time ago?
Asked on Mar 07 2012
Thank you for providing me with this extra information. It has allowed me to more fully understand the problems you have been experiencing with ab49873 and ab93434 previously.
Having looked at the protocol I have a few further questions:
1. Exactly how was the tissue fixed? For how long was the tissue treated with PFA? What concentration of PFA was used?
2. What were the samples tested? Which species did the tissue come from?
3. Had the goat anti rabbit used for both of the experiments been used successfully before?
As ab93434 has as yet to our knowledge not been used in staining frozen sections it may be that the antibody is not suitable for this application. However, there are a few suggestions I can make which may help toimprove the results seen so far.
1. I would suggest decreasing the dilution of both the antibodies. Although ab49873 is recommended to be used at 1/5000, this is only a starting point and may need to be optimised depending on the tissue and protocol used. With ab49873, I would suggest initially trying 1/500 to see if there is any signal. With ab93434 we recommend using 5 µg/ml as an initial starting point, I would therefore use a dilution of 1/50 to start.
2. As you have not been observing any staining with overnight incubation at 4 degrees, I would try incubating at room temperature for 2 hours to see if this improves the results.
3. I would increase the level of Triton X used to 0.2 % for performing the permiabilisation.
I hope these protocol tips are of use, with the answers to the questions above I may be able to provide further suggestions.
I am able to send you ab91073 directly as I suggested to detect theL isoform of glutaminase as a good-will gesture for the bad experience you have had. If you would prefer to change this to ab60709 I can also organise this.
I look forward to receiving your reply.
Answered on Mar 07 2012