I am trying to perform a Duolink (Proximity Ligation Assay, Sigma) assay on freeze-mounted rat brain sections - so far without sucess - and am wondering if someone has some insider-knowledge to share.
I have tested the antibodies and the Duolink kit in cell culture and got a beautiful positive response.
I also optimized the antibody staining of the sections for immunefluorescence. They are paraformaldehyde fixed, freeze-mounted, microwaved in citrate buffer for antigen retrieval, permeabilized with triton for 1 h, and blocked for 1 h with horse serum/BSA/PBS
Now, the only thing that still doesnt work is the combination of the two protocols.
Who has successfully done Duolink on PFA-fixed and freeze-mounted sections?
Thanks for your input!
Hi,
I never used the system but is it possible to test both primary antibodies separately (in a classical way) to see which one is the problem? If the DuoLink works in cell culture (means unfixed cells): Have you tried unfixed sections? And last but not least: Are you sure, that your target proteins are close enough? Maybe that's already the answer to your question: If the controls are successful and the proximity ligation assay is not, it might want to tell you, that your proteins are not close together.
Hi Vivica,
Thanks for your suggestions. The assay works in fixed cultured cells with two antibodies directed against the same protein. These antibodies also give a nice immunefluorescent staining in the sections. I think I will splash out on ordering a new kit, and change incubation times.
I wish you good luck, that it works with a new kit, but since your controls are as they are supposed to be (DuoLink works in general (tested on the cells), your antibodies bind to sections, you don't have a technical problem in the detection) I doubt that you will see what you want to see... Negative reaction means that your proteins are not close to each other. To be sure, you should think about a control pair of antibodies that are definitely close enough to react with DuoLink on a section (search in literature).
Hi Margarethe, I was wondering if you were able to get the Proximirt Ligation Assay PLA) to work on brain section? and If you have a protocol for that ! I have the same issue, the PLA worked nicely in cell culture, but not on brain section. The main issue I have with the brain section is very high backgrounds that interfere with the PLA signals. I would appreciate any suggestions. Thanks
Hi Amina,
I doubled all incubation times. That gave a signal, however, it has high background, as you say. My test condition is reproducibly 2x higher than the negative controls (I have two negative, one with unrelated antibodies, one without), so for my purposes its enough, but its not very pretty. The positive control is 10x higher than negative control, so that is actually quite nice. I use a different blocking buffer than in the cell culture: Roche Blocking Solution. Its meant for ISH, but I use it for Immunefluorescence as well. In cell culture I use BSA/Horse serum.
Hi Margarethe,
I have extensive expertise in proximity ligation assay (PLA) in heterologous cells, mice tissue (both perfused and postfixed in PFA 4%) and human tissue (FFPE slides). We recently published a protocol (https://www.ncbi.nlm.nih.gov/pubmed/27960030). In the manuscript you can also find postimaging analysis (deconvolution and dots counting), tips and tricks. Also, check this webinar... https://event.on24.com/eventRegistration/EventLobbyServlet?target=lobby20.jsp&eventid=1263038&sessionid=1&key=1A3636EF49A71AA40D2A5061EE1757F8&eventuserid=154096643
Let me know if you need further assistance,
Best wishes!
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