My previous question regarded antibody selection against BrdU for detection of proliferating cells in the sub-granular zone of the dentate gyrus in mice. Fortunately, the protocol passed down to me using a mouse-host antibody from BD and included DAB and cresyl violet counterstained worked (to my delight) quite nicely --- see attached image. This protocol was for bright field microscopy.
I am interested now in an immunohistochemistry protocol that involves co-labeling of BrdU and the neuronal cell marker NeuN for fluorescence microscopy to determine the proportion of cells that are per se neuronally fated in mouse dentate gyrus. Protocols using triple-labeling for GFAP would also be useful. Any insights into this technique would be greatly appreciated.
Thanks in advance, and please feel free to message me questions on study design, etc. if it helps to better answer technique-related questions mentioned above.
Hi Steven, how are you doing?
Maybe I can help you.
I used this follow protocol for hippocampus slices
Look this: In short, the sections were rinsed three times and left
overnight in 0.1 M PBS to remove the antifreeze solution. The sections were
then placed in boiling citric acid (0.01 M, pH=6.0) for 7 min, rinsed 5 times with
PBS for a total time of about 20 min, incubated in 2.4N HCl at 37 °C dissolved
in PBS for 30 min, and rinsed 5 times with PBS for a total time of about 25-30
min. Afterwards, the sections were incubated in mouse anti-BrdU ,
Novocastra Laboratories) in PBS (1:200) containing 0.5% Tween 20 for 48 h.
The sections were washed 5 times with PBS for a total time of
about 25-30 min, and then incubated for 4 h with Alexa Fluor® 488-conjugated
Neuronal Nuclei (NeuN) mouse monoclonal antibody (Millipore, 1:100)
So, hope to hear from you as soon!
Hi Steven, how are you doing?
Maybe I can help you.
I used this follow protocol for hippocampus slices
Look this: In short, the sections were rinsed three times and left
overnight in 0.1 M PBS to remove the antifreeze solution. The sections were
then placed in boiling citric acid (0.01 M, pH=6.0) for 7 min, rinsed 5 times with
PBS for a total time of about 20 min, incubated in 2.4N HCl at 37 °C dissolved
in PBS for 30 min, and rinsed 5 times with PBS for a total time of about 25-30
min. Afterwards, the sections were incubated in mouse anti-BrdU ,
Novocastra Laboratories) in PBS (1:200) containing 0.5% Tween 20 for 48 h.
The sections were washed 5 times with PBS for a total time of
about 25-30 min, and then incubated for 4 h with Alexa Fluor® 488-conjugated
Neuronal Nuclei (NeuN) mouse monoclonal antibody (Millipore, 1:100)
So, hope to hear from you as soon!
Hi Livia,
Thanks very much for responding. This protocol looks to fit our needs well --- is there published work that uses this? It would be nice to have literature support.
I don't suspect this will be a problem, but we have been slicing brains on a vibratome and conducting slide-based immunohistochemistry (as opposed to using free-floating sections) --- do you think there's reason to believe this will be a problem with the co-labeling protocol?
Also, is it normal to not have a secondary antibody incubation step in the co-labeling protocol? I suspected there would have to be an incubation in secondary antibody from the non-host species (e.g., horse anti-mouse). Perhaps this step is only necessary for bright-field microscopy procedures.
Is there any last step necessary before coverslipping after the tissue has incubated in NeuN primary?
If you have this protocol in a Microsoft Word document or PDF and feel you are able to email it to me, that would be very greatly appreciated --- but I understand if not! Again, thank you for getting back to me, and I look forward to hearing more about this protocol and its potential utility in our lab!
My best,
SS
Hi Steven, sorry for not replying sooner! Look for Barry L Jacobs protocols, we adapted this protocol because we use slide-based immunohistochemistry., it might work! We use NeuN conjugated to avoid another secondary antibody step. Another last step before coverslipping is not necessary, just wait about 12h to read the slice! I can send you the full protocol, please let me know your email adress. with my best Lívia
That would be fantastic, Livia, thank you very much. My email address is [email protected].
I am familiar with Barry Jacobs and his colleague Liz Gould over at Princeton --- I always love connecting the dots between researchers. Our other protocol was adapted from Tracey Shors' lab, where I believe Liz Gould spent time as a post-doc. Anyway, I would appreciate the protocol, and I'll do some searching through Barry's work to find support for using the protocol. Also, I'll be sure to send questions your way on protocol specifics when they inevitably arise :)
Again, thanks very much for taking the time to help me!
Hi Steven, I am a graduate student from Binghamton University and our lab just had an accepted manuscript that used triple-immunofluroescent staining that describes the concentrations needed and also the suppliers. We looked at BrdU co-localization with NeuN, GFAP, Iba-1 and NG2 in 2 stains. If you click on my profile I uploaded the full text of the accepted manuscript on cytogenesis as a response to exercise on there. If you have any questions you can feel free to send me a message on any of the technicalities or the precise protocol. My e-mail is [email protected]. I realize getting staining protocols up and going can be an arduous process in the beginning...
Hi, everyone. Thank you all for giving your input on this question. My small lab is also looking to stain some rat brain sections we are about to cut. The rats were injected with BrdU. We will look in hippocampus and olfactory bulb to see whether neurogenesis was affected by the diets we fed animals. Do you all have any recommendations for a protocol that might work for both? I would love to do slide based instead of free-floating if we could.
Thanks!
D.
Hi Darryl! We use slide-based immunohistochemistry, the brain slice fall during antigen retrieval step with citric acid and HCL, but we use plus gold electrostatic slices to solve this issue. Antigen retrieval time its critical also. My protocol work for both areas. If you have any questions you can feel free to send me a message on any of the technicalities or the precise protocol. My e-mail is [email protected].
My best
Hi, everyone. I have posted my own question (and got no answer yet) but while I was seeking information to resolve my problem, I found this question/answers so I thought that you will probably be able to help me since this topic is about the same labelling I am trying to do. It is double labelling of BrdU and NeuN (free-floatng sections) and lately I have had trouble with NeuN in the dentate gyrus. I get a very nice labelling in cortex but very few cells label in the DG where it used to do so very well too. I use MAB377B with Streptavidin-488. I use TBS as buffer and block with the blocking buffer from TSA amplification kit since I amplificate BrdU signal (Cy3). I do a pre treatment with HCl and I have tried citric acid without getting any better results (but it used to work even without the citric acid step).
Thanks in advance!
@mariebedos I used to have problems with NeuN as well, to solve this I started to used NeuN conjugated to Alexa 488. Sometimes I need to make HCl and citric acid. And also the perfusion with PFA 4,% with PB helps. Feel free to contact me on [email protected]. hope it can help you. Best
Thank you for your response Livia unfortunately I don’t think my lab can afford to buy the conjugated antibody :(
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