Anti-pan Cytokeratin antibody [PCK-26] (ab6401)

Here you are my answers to abcam in details regarding my project so please forward to them and hopfuly I can get their guide asap. I am isolating human hertwig's epithelial root sheath (HERS) cells from periodontal ligament (PDL). So the cells that I am isolating are epithelial that should express these two epithelial markers as published in the literature (E-cadherin and Pan-cytokeratin) and will compare them with Stem cells from Human exfoliated deciduous teeth (SHED) using another two anibodies (CD44) that should be highly expressed in SHED than HERS and (CD73) that will be expressed similarly in both cells. So for characterization I ordered these antibodies from abcam: 1-      Mouse monoclonal (HECD-1) to E Cadherin (ab1416) 2-      Mouse monoclonal (PCK-26) to pan Cytokeratin (ab6401) 3-      Mouse monoclonal (F10-44-2) to CD44 (ab6124) 4-      Mouse monoclonal (7G2) to CD73 (ab54217) -          Immunofluorescence staining steps (first time) HERS were cultured in 4 well culture slide and the second day we did the following: a-      Discard the medium b-     Wash 3 times with PBS each time two mins c-      Add 500 µL methanol to each well for 20 mins at 4°C d-     Discard methanol and wash 3 times with PBS each time two mins e-      Add 7 drops of blocking reagent (from Millipore/ comes in the kit of immunoperoxidase secondary detection system) keep it at room temperature for 1 hr. f-       Discard blocking reagent and add the primary antibodies (1:50 ratio) (each antibody was applied separately in each well of the same chamber slide) (we diluted the antibodies using PBS) g-      Keep the chamber slide in 4°C for overnight. h-     Then discard the antibodies, wash 3 times with PBS each time two mins i-        Add 500 µL of secondary antibody (anti mouse FITC) to each well and keep it for 1 hr at room temperature j-       Discard secondary antibody and wash 3 times with PBS each time two mins k-     Add 500 µL of DAPI to each well keep it for 1 hr in a dark room l-        Discard DAPI and wash properly with PBS m-   The cover of the chamber slide was chopped and fluorescence mounting medium was applied (from Dako: S302380) n-     Cover slip was placed and the observed under fluorescence microscope image analyzer Under microscope: We observed the following (as I sent you previously some images); Green cells with green background and blues nucleus. So how to get black background with only green cells and blue nucleus as we got used for immunofluoescence images? -          We did optimize the method (second time) using the same previous procedures but with primary antibodies 1:300 but we got the same result. -          We did optimize the method (third time) using the same previous steps but with these changes: a-      We used PBS with Tween-20 for proper washing b-     Apply methanol and keep it in -20°C for 5 mins c-      Primary antibodies were applied with ratio (1:500) and keep it for 9 hrs at 4°C d-     DAPI was applied for half hr in a dark room. -          But under microscope we got the same result. So we are planning to use primary antibodies 1:1000 and keep it for 3 hrs but we do really need your recommendation and help to get the correct result before we run the test. And waiting for your new quotation please as I did email you. Best regards,    

ANSWER:

Thank you for contacting us.

The background should be black. I am sorry we never had a question like this before. It looks like this problem might be due to instrumentation then antibodies itself.

I have following recommendations that might help; - Use less number of cells per well. - Check the viability and passage level of cells. - The cells must be healthy with low number of passages. - Avoid drying out plate between washings. - Check microscope settings. - Check if the blocking is sufficiently done. You can also use 3-5% BSA for 1-2 hour in PBST for blocking. - Use more diluted primary antibodies.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

I have some technical questions about the Ab against pan-Cytokeratin PCK-26 (ab6401-100). Could you give me the info on the recommended fixation and dilution for immunocytochem and the Ab source (eg. cultured supernatant, ascites)? Your response will be greatly appreciated.

ANSWER:

The antibody is ascites. We recommend the standard procedure listed on the website for fixation and dilution. No special requirements are necessary since this antibody appears quite versatile and has been used in many applications successfully.

Does this antibody stain the dermal layer as well as the epidermis?

ANSWER:

Sorry, we do not have any data on this.

Would this antibody differentiate epithelial cells form estromal cells in rat mammary gland by western blot?

ANSWER:

Sorry, we do not have any data about the use of this antibody in this system.

I am trying to stain cytokeratin-positive cells from digested murine tumour tissue for analysis on flow cytometry.

I have tried other pancytokeratin and human epithelial antigen antibodies from DAKO. Altough they cross-react with murine cells in vitro (tumour cell lines), they don't label them when grown in mice.

Can you help me here? I am trying to assess tumour burden in primary tumours.

ANSWER:

This antibody also stains murine cells in vitro, but unfortunately we have no data on the use of this antibody on tumours. You may find an answer to your question in ref.2 on the datasheet.