Pancreatic Duodenal Homeobox1 protein is a intranuclear protein and I need to stain for it with an antibody. I have used Abcam and RD antibodies and their not working, i have changed the permeabilization states as well as the concentrations for the antibody, has anyone used PDX1 in mice and if so what concentration and the protocol? The sections I'm using are mouse pancreatic tissue. Help please!!
Dear Qadir,
It would be easier to troubleshoot if you share details about your antibodies. How do you know that it is not working? Are you not getting any signal during scan?What was the thickness of your tissue section? What was the dilution factor for your primary and secondary antibody? What type of conjugated secondary antibody have you used ( Cy3, FITC or Alexa Flour)? For me, I use to go beyond 1: 50000 dilution for primary Ab and 1 : 100000 for secondary Ab.
You can go through the links
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections
http://www.abcam.com/ps/pdf/protocols/double%20immunofluorescence%20-simultaneous%20protocol.pdf
Hope for the best.
Hello
i have good expression when used PDX-1 to express mouse pancreatic stem cells by IHC technin , you can get it in this link :
https://www.researchgate.net/publication/280924090_Expression_Pattern_of_Stem_Cell_Markers_in_Developing_Mouse_Pancrea
and look at link about "Protocols and troubleshooting tips " may help you
Good Luck
http://www.abcam.com/index.html?pageconfig=popular_protocols
Article Expression Pattern of Stem Cell Markers in Developing Mouse Pancreas
Dear Qadir,
i came across same problem with u， though i follow the protocol ···For me, I use to go 1：200 dilution for primary Ab and 1 : 500 for secondary Ab（abcam)and the thickness of tissue section is 6-10um.Both paraffin section and frozen section was used in experiment ,but pdx1 can not detect by immunofluorence....
Have u solve your problem?? Help please~!thx·~~
http://www.abcam.com/ps/pdf/protocols/double%20immunofluorescence%20-simultaneous%20protocol
Thank you everyone for your input, I have been able to successfully stain with PDX1 in Parrafin sections of human pancreas. Ranging from 5-10um in thickness.This is for IF.
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