Hello Yirou Zhang,

To confirm that your primary antibody is working, include a positive control in your experiment.

For immunofluorescent staining, you might use a tissue or cells known to express the protein of interest. Choose a sample where the target protein is known to be expressed at high levels. A positive signal in the positive control confirms that the primary antibody and your experimental setup are working correctly. 

Simultaneously, to show that the primary antibody does not bind non-specifically to other cellular components, choose a sample that does not express the target protein. If that is not possible, then a sample having low expression can be used. You should try a negative control because no or very weak signal in the negative control will indicate that the primary antibody is binding specifically to its target. 

So, by carefully including a positive and a negative control and after you have optimized your antibody dilution, you can be confident that your primary antibody is working correctly and that your results are reliable. 

Best,

Hi Yirou,

in addition to what Malcolm Nobre is advicing (which sounds very convincing to me) for testing the functionality of your antibody I wanted to say, that for a lot of antibodies repeated freeze and thaw cycles should be avoided, as this can lead to reduced antibody functionalty and if you are unlucky even to signal loss (depends also on the antibody and the buffer used).

For most antibodies it is recommend that for longtime storage you aliquot the antibodies and store the aliquots at -20°C. Short time they can be often stored at 4°C.

If you are not sure about the storage conditions I would contact the provider of the antibody (if possible).

You can also find a nice summary regarding storage conditions here:

https://www.celnovte.com/solution/how-temperature-affects-primary-antibody-stability-in-western-blotting/

For your frozen aliquots you could consider adding glycerol to a final concentration of 50% glycerol (acs grade glycerol). This will keep it them freezing/cryoprotect the aliquots.