Hello every body, I am going to use Numa and Tubulin to detect the cell divisions ( almost during meta-phase stage) in mice skin I tried
alpha Tubulin Monoclonal Antibody (TU-01) as a tubule markers
and Anti-NuMA antibody (ab84680) as a centriloe markers
I got something a weak result specially with Numa
Could any one kindly help me in the good antibodies used in mice samples and what is the best method used for this
Thanks in Advance
Best Regards
Ayman
Here are a few reviews of anti-Tubulin Abs: http://pabmabs.com/wordpress/?cat=468 that might be worth reading. If no one here gives you specific recommendations, we can offer a number of possible sources for you to explore (e.g. https://www.linscottsdirectory.com/antibodies?q=numa). Filter for "mouse" reactivity and follow "more info" to suppliers' data pages.
Here are two mouse antibodies from Sigma that I have used with links to the Sigma website.
α-Tubulin T6557(Spindle fibers)
https://www.sigmaaldrich.com/catalog/product/sigma/t6557?lang=en®ion=US
γ-Tubulin T5326 (Centrosomes)
https://www.sigmaaldrich.com/catalog/product/sigma/t5326?lang=en®ion=US
If you signal is week you can incubate the antibodies O/N @ 4° C. Also, after fixing your slides with 4% paraformaldehyde, you can pretreat you slides in methanol for 2-3 minutes prior to applying antibody.
Attached is a protocol that I have used with success.
Did you ntry it before?
and I didnt see any secondary antibody in the protocol
Isnt there ant secondary antibody?
Also what is the role of (antibody blocking solution)
is it similar to Antigen unmasking solution?
also what is the appropirate Numa antibody we can use ?
If you look at step 7 of the protocol you will see that it is the secondary antibody step. You can, of course use a secondary antibody with any fluorescent tag you like. Antibodies that are already tagged with a fluorescent molecule are also available. These do not require a secondary antibody. However, if you are having problems with a weak signal, the protocol I provided would be a good place to start.
I have had great success with Alexa tagged antibodies from Molecular Probes (now part of Invitrogen). Here is a link to the Alexa488 antibody (this is a green fluorescent molecule that can be used with a FITC filter)
https://www.thermofisher.com/antibody/product/Goat-anti-Mouse-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11001
The antibody blocking solution prevents nonspecific background and is very important. There are many variations of this solution but this one has worked well for me.
I am not familiar with antigen unmasking solution so cannot offer any help with that question. I also not used Numa either so am not certain what would be best for you.
Hope this helps.
Leslie