Hi there,
I'm optimising a protocol for BrdU/NeuN co-localisation on 40um mouse brain sections of the hippocampus on slides. I previously got positive signal with both antibodies on their own but have run into trouble when I have attempted to co-stain them.
I'm now considering sequential staining for each antigen (NeuN first followed by BrdU - or should it be the other way around?) For sequential staining in this fashion, would it be necessary to fix the first antigen with PFA for instance before proceeding onto the second stage of staining?
Alternatively, i have seen several protocols which have used a longer incubation with the BrdU/NeuN primaries of 48 h.
My current protocol is as follows:
3x5min TBS, 2N HCl for 30min at 37C, 2x5min with borate buffer, 2h block with 10% goat serum in 0.2% TBS-T at RT. Antibodies used were NeuN Ms (Millipore) @ 1:200 and BrdU Chk (Abcam) @1:100 in 1% goat serum o/n at 4C.
Day 2 is 3x5min TBS, Ms 488 and Chk 633 Alexa Fluor @ 1:1000 in 1% serum for 2h at RT, 3x5min TBS, DAPI (1:500) for 5min, 2x5min TBS and coverslip with Vectashield.
Comments or discussion on this are greatly welcomed!
Thanks in advance
Sinéad
Hi Sinead,
I do the always the sequential staining. First incubation ON with NeuN (the same as you 1:100), the day after postfixaxion with 4% PFA (30' RT is enough) and then HCL treatment (I always do 25' 37C), then washing with PBS, blocking again and then BrdU incubation ON (I use the rat Ab from abcam, I do not know about the Chk) and the day after secondaries for both.
If you put both together and then do the HCl without fixing the antigens get destroyed.
I hope it helps :)
Hey Sinead,
We have a working protocol here in the lab using BrdU from abcam and NeuN from Millipore. It is sequential protocol with BrdU incubation on day 1 and NeuN on day 2. We use PBS instead of TBS, do HCL incubation for 45 mins and subsequent neutralization with sodium tetraborate two times for 10 minutes. We found the preparation of the buffer crucial for the success of the protocol. I can e-mail you a more detailed protocol if you like.
Thank you all for the suggestions. I will try a sequential staining protocol on the basis of some of your suggestions.
Sara, I was interested that you wait until the end of the protocol and both secondaries simultaneously, I would have thought to do the NeuN primary and secondary and then start the BrdU protocol.
Danka, nice to hear from you and thanks for the offer of the protocol. I'll give it a go myself first and if I'm still have problems I'll drop you an e-mail.
I also have the same kind of issue as you. I would like to co-stain with GFAP and Iba-1 and DCX instead. But want to know if you successfully stained it or not?
Danka, can I talk to you directly about BrdU??Thanks a lot!
Louis
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