Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326)

Does this antibody recognize Edu?

ANSWER:

Thank you for contacting Abcam.

The Abreview that shows reactivity with Edu can be found at the link below:

http://www.abcam.com/index.html?datasheet=6326&tab=abreviews&intabreviewid=25172

Please let me know if there is anything else I can help you with.

Product code: 6326
Inquiry: Kindly advise if ab6326 will be covered by Abpromise for the staining of isolated DNA fibers according to the protocol below. In the past the customer used to work with the same clone of Brdu antibody from xxxxx and it worked well. Recently, they changed something in the production (different supplied volume and another dilution should be used) and the customer wishes to switch to Abcam's alternative. Here is the protocol:
Replication detection Isolated DNA fibers are stretched on silanized slides before replication detection.
Slides 1) Denature in 0.5M NaOH / 1M NaCl for 10 min (slow/minimal agitation) (In 50ml tube mix: 20ml 1M NaOH + 20ml 2M NaCl) 2) Wash three times quickly in PBS 1X 3) Wash two min in 0.5 M Tris HCl pH 7.5 / 1M NaCl (In 50ml tube mix: 20ml 1M Tris HCl pH 7.5 + 20ml 2M NaCl) 4) Wash three times quickly in PBS1X 5) Dry with a kimwipe ready for antibody staining Use “simple” glass coverslips for all the procedure Replication detection (No hybridization) Use undiluted Blockaid from molecular probes as the detection buffer throughout (The stock is kept in aliquots of 500μl at -20°C. This blocking solution is very good for reducing the background). Use 25μl of mixed antibodies for each slide. All steps at 37°C. Incubation times: 1st Ab 45-60min 2nd Ab 25-40min. Wash 3X 3-5 min PBS1X after each step with gentle agitation at RT. Don’t vortex antibodies. Mix by pipetting. Replication IdU ReplicationCldU mBrdU (5/25) rBrdU (1/25) gm AlexaFluor 488 (1/25) gr AlexaFluor 594 (1/25) 1st 2nd Dil X2 X3 X4 X5 Dil X2 X3 X4 X5 mBrdU 1/5 10 15 20 25 gm488 1/25 2 3 4 5 rCldU 1/25 2 3 4 5 gr 594 1/25 2 3 4 5 BA 38 57 76 95 BA 46 69 92 115 DNA detection Use 25μl of diluted (1:25) anti-ssDNA antibody in BA for each slide. Use 25μl of diluted (1:25) 2nd antibody goat anti-mouse Alexa Flour 350. Wash 3X 3-5 min PBS1X after each step with gentle agitation at RT. Incubation times: 1st Ab 45-60min at 37°C 2nd Ab 25-40min at 37°C. Slides should be dried at RT. Mount in Slowfade Light from molecular probes. Thanks in advance for your assistance and repy. Have a nice day.

ANSWER:

Thank you for contacting us.
Unfortunately, we have not tested the antibody in such an application and can therefore not guarantee that the antibody will work. Although as your customer has used this clone before, it is very likely to.
As this is not currently a supported application I would not normally be able to offer a testing discount either. But if your customer would be willing to provide me with the protocol used and the results obtained (in the form of an image) I can offer a testing discount if they would be interested.
If you would like for me to generate the testing discount code please do let me know. I would need the customers name and institution.
I look forward to receiving your reply.

Optimizing the protocol to stain for BrdU, Sox2, and Oct4 on the same sections.

ANSWER:

Thank you again for your call last week and for your patience while I have looked for some more information for you.

I wasn't able to find any helpful suggestions in the literature, but I did find the following post on an IHC message board:

http://www.ihcworld.com/smf/index.php?topic=998.0

It does look like others have stained for one antigen, fixed the tissue, then stained for BrdU. You might also be interested in posting a similar post on this message board to see if others have any more suggestions.

Please keep me updated about your progress, and let me know if youhave any questions or if there is anything else that we can do for you.

Optimizing the protocol to stain for BrdU, Sox2, and Oct4 on the same sections.

ANSWER:

Thanks for your call today. It was a pleasure speaking to you.

I will look through some literature and see if I can find any additional suggestions about performing the sequential stainings. These are the Sox2 and Oct4 antibodies that I mentioned which might work better following heat denaturation:

http://www.abcam.com/Oct4-antibody-ChIP-Grade-ab19857.html

http://www.abcam.com/SOX2-antibody-ab97959.html

I will be in touch once I've finished with the lit search. Please let me know if you have any questions or need anything else in the meantime, and I'll be happy to help. Have a great weekend!

BrdU staining worked well, then added DAPI and the signal was quenched within a couple of hours. AF488 secondary antibody. DNA was denatured with citrate based buffer in microwave for 2.5 min, slides warmed for 5 minutes. Saw auto-fluorescence from crystallized DAPI then washed with DI water, put cover slip back on slide. This has never happened before. DAPI signal looks fine.

ANSWER:

Thank you for your call yesterday and for your patience while I have looked into this situation.

I believe you mentioned that you would be performing the staining again today- have you found any new results?

I'm still not exactly sure how to explain this situation, but I do have a couple of ideas. First, I would recommend just staining for BrdU without the DAPI to see if the signal is quenched even without the DAPI. If the quenching only occurs upon staining with DAPI, you could try usinga different DNA dye like DRAQ5 (ab108410) though I think the problem might persist regardless of which dye you choose. If the quenching occurs even without DAPI, then make sure you're following all steps to avoid photo-bleaching. Finally, as we discussed, if all else fails then you can image the slides during the first couple of hours before the AF488 signal is quenched.

I am sorry that I can not be of more help at this time, but please keep me updated with any progress and let me know if you have any questions. I look forward to hearing from you.