Anti-Calbindin antibody [CB-955] (ab82812)

Inquiry: Dear Abcam. I am writing you for having some more information about the antibody ab82812 (anti-calbindin). I used it in rat brain perfused in PFA 4% for IF as a neuronal marker in the the deep cerebellar nuclei. I got good results but a Referee raised the question that it cannot be used as a specific neuronal marker but just a marker for Purkinje cells. I definitely disagree with him! Your datasheet is pretty clear about the use of this ab as neuronal marker and caldibin is commonly used in this way (...). What do you think about? Do you suggest me a possible reply? Thank you for your time and your attention. Kind regards,

ANSWER:

Thank you for contacting us.

I am glad you have found the anti-Calbindin antibody [CB-955] (ab82812) of use in staining your rat brain tissue.

I, like you, have found several references where Calbindin has been used as a neuronal marker. I include a sample below. I am sorry that the referee of your paper does not support this. I would suggest doing a comprehensivejournal search to find prominent papers where anti-Calbindin antibodies have been used for this purpose and present this to the referee.

I am sorry that I could not be of more help in this instance. I wish you all the best with your publication and your future research.

References whereCalbindin has been used as a neuronal marker:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0032689

Hello, Can you tell me what type of permeabilization you used at the 1/15 and 1/30 primary concentrations and what the conditions (time, temperature) of the primary incubation were? I am still not having success with this antibody. Thank you

ANSWER:

Thank you for your reply. From what I can find out, ab82812 was tested with a 30 minute primary antibody incubation period at room temperature and there is no notes about whether a permeabilzation step was used or not. We do have multiply other Calbindin antibodies in our catalogue and I have looked through the Abreviews for these products and have found some information that may be helpful. From Abreview for ab49889: IHC-Fr Sample Mouse Tissue sections (Brain section) Specification Brain section Fixative: Paraformaldehyde Permeabilization: Yes - 0.3% TritonX in 0.1% PBS Blocking step: Serum as blocking agent for 1 hour · Concentration: 10 % · Temperature: 24°C Other product details Dilution 1/200 Incubation time: 24 hours Temperature: 4°C Secondary antibody Name: Non-Abcam antibody was used: anti-rabbitAlexa 488 Conjugation: Alexa Fluor® 488 Dilution: 1/1000 From Abreview for ab114266: IHC-P Sample Human Tissue sections (brain) Specification brain Fixative: Formaldehyde Antigen retrieval step: Heat mediated - Buffer/Enzyme Used: EDTA, pH8, 20 min, 100C Permeabilization: No Other product details Dilution 1/5000 Incubation time: 20 minutes Temperature: 25°C Diluent: Dako diluent, background reducers Secondary antibody Name: Non-Abcam antibody was used: Goat anti-mouse anti-rabbit IgG, Leica Refine Conjugation: HRP polymer As I said the above information if from Abreviews submitted by customers who used other Calbindin antibodies successfully and maybe some of that information will prove useful in getting ab82812 to work in IHC-Frozen Sections. Please let me know if there is anything else I can help you with.

 I am trying to use ab82812 (mouse monoclonal to calbinin) to label neurons in embryonic rat brainstem. I am using free floating paraformaldehyde fixed 100 micron sections. I have tried 24 hr incubations at 1:50, 1:100 and 1:200 at room temperature and at 4C followed by a secondary reaction with Rhodamine conjugated sheep anti-mouse Rhodamine (2hr room temperature) and I am not seeing any positive results. Do you have a protocol you suggest for immunofluoresence with this antibody?

ANSWER:

Thank you for contacting Abcam.

We have not tested ab82812 in free floating sections, so I am unable to send you a specific protocol for doing Free Floating section staining. However when using paraffin embedded PFA fixed slides, it is advised to use the following method:

1 - Use antigen retrieval, 10mM citrate buffer, pH6.0.

2 - Primary antibody concentrations between 1/15 -1/30 dilutions.

I hope that the above protocol information proves to be helpful. If there is anything else I can help you with, please let m