In my lab we generally used to do immunocytochemistry regularly but now its not working that means it does not show any signals after secondary antibody addition with background or almost no stain red . what could be the reason?
Thanks chen actully i used new primary antibody and secondary one was brought 2 year before which was kept in -80C and is there any possibility that secondary antibody could have died due to long term preservation in -80....
Dear Habib,
First you have not given any protocol you used. Did you aliquot antibody when it was purchased. Or you have been thawing and freezing the same antibody again and again and again. If that is true, than your antibodies may have got bad. Too many times thawing freezing is not good. When I buy antibody, I aliquot in many tubes and freeze them at -20C or 4C as instructed by manufacturer (mostly at -20C). I take one aliquot and use it in one or 2 assays without multiple freezing.
Mostly likely, you might have frozen, thawed multiple times, than antibodies will not work.
If you give more details of steps I can help you,
Best luck,
Subhash
Dear Nazmul,
I addition to comments above, All antibodies (primary or secondary) are vulnerable and are prone to damage. Since you have tried a new antibody, it is more likely that your secondary is damaged. Saying that you shoudl also check if your primary is from the same batch as your previous working antibody. Sometimes an antibody from the same company does not neceassarily give you the same results, especially if they are originating from different batches, meaning produced in a different host animal (eg, rabbit). There were similar discussions at RG in the past, see below
Best wishes,
Refik
Technical Report Research Methods and Technical Considerations: Answers to ov...
Hi Nazmul,
Yes long-term storage and repeated thawing will damage the antibody. Please try using a positive control as suggested by Hyde Chen to confirm this. You may use a different primary antibody that you know is working with the secondary antibody that you described in this post. Another control will be using a different secondary antibody for the primary antibody you just described in the post.
All the best.
Hope you figure this out!
You do realize that you are using the least sensitive of all immunocytochemical techniques. Thus even a small loss of antibody titer, antigen reactivity etc will reduce detection. If you follow our protocols, you can likely resurrect tired antibodies. It is important to leave the antibody on the tissue for 48 hrs as it will increase the sensitivity 3-4 fold. (using TSA amplified fluorescence will make the antibody much better (more product with 100 fold less antibody). Freezing and thawing in my 40 years of experience has only affected a small subset of the >500 antibodies we've tested. Santa Cruz antibodies were the most likely to fail upon any storage. Keeping them at -20˚ can cause sublimation; -80˚ is better. If your antibodies had low titers (stainining at >1:5000 with ABC peroxidase staining), then stability is a greater problem - dilute antibodies are not very stable and the companies frequently over dilute them. Our paper attached gives you the conversion when shifting methods. Sometimes how tissue is stored prior to staining can be a factor. frozen/paraffin sections can lose reactivity. Storing freely floating sections gives the greatest maintenance of immunoreactivity (using antifreeze solutions and maintaning sections at -20˚. They keep that way for >25 years).
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