Liver in which species?

Liver of rats

Hello Khaled! I use the antibody for ck19 from Novocastra I do not use any special protocol. Just dewaxing is performed (if paraffin sections) and then antigen retrieval with citrate buffer pH 6 (10 minutes x2). In human livers. 

I asked because I have protocol for mouse CK19. It uses anti CK19 rat antibody, which will not be useful for you.

Immunohistochemistry Using Slide-mounted Paraffin Sections

All steps in the following protocol are carried out at room temperature unless stated otherwise.

Recipes for solutions highlighted bold are provided at the end of the protocol.

Deparaffinizing and rehydration

  1. Immerse slides in xylene for 10 minutes. Repeat this step again in fresh xylene         for 10 minutes.

       (If required, repeat a third time in fresh xylene for another 10 minutes.)

  2. Rehydrate sections by sequentially incubating with 100%, 95%, 80% and 60%           ethanol for 5 minutes each.

  3. Rinse sections with distilled water three times for 3 minutes each.

      Antigen retrieval (optional)

  4. Transfer slides to a microwave-proof container and cover with citrate buffer.

  5. Heat in the microwave on medium power for 10 minutes.

  6. Allow slides to cool in the citrate buffer for approximately 35 minutes.

      Primary antibody incubation

  7. Rinse slides three times with 1X TBS for 3 minutes each.

  8. Incubate slides with 3% H2O2 solution (diluted in distilled water) for 10                     minutes      to quench endogenous peroxidase activity.

  9. Rinse slides three times with 1X TBS for 3 minutes each.

10. Prepare 5% normal blocking serum in 1X TBS. The serum should be derived             from the same species in which the secondary antibody was raised. Block the         sections for 1 hour. (Alternatively, use 5% BSA in 1X PBS for blocking if the                 corresponding serum is not available.)

11. Incubate sections with primary antibody diluted in 1X TBS for 1 hour, or                       overnight at 4°C; the optimal antibody dilution ratio should be pre-determined            by experimentation. Set up negative controls by omitting the primary antibody            incubation step for one slide per each experimental condition.

12. Following primary antibody incubation rinse slides three times with 1X TBS for          3 minutes each.

    Signal Detection

     (Proteintech routinely uses EnVision Kit reagents (Dako) for this step.)

13. Apply sufficient peroxidase labeled polymer and incubate for 30 minutes.

14. Rinse slides three times with 1X TBS for 3 minutes each.

15. Prepare an appropriate volume of substrate solution prior to use by mixing one        drop of Liquid DAB plus chromogen immediately with 1 ml of substrate buffer.         Apply the substrate carefully and incubate for 5-10 minutes till a brown color             develops.

16. Rinse sections gently with sufficient distilled water.

       Mayer Hematoxylin counterstaining (optional)

17. To stain nuclei, immerse slides in a bath of hematoxylin for 3 minutes.

18. Rinse slides gently with distilled water.

19. Transfer slides into a 1% HCl, 99% ethanol solution for 10 seconds; transfer to        distilled water immediately.

       Dehydration and mounting

20. Immerse slides sequentially into 60%, 80%, 95% and 100% ethanol baths for          5 minutes each.

21. Immerse slides in xylene for 5 minutes. Repeat this step again in fresh xylene          for 5 minutes.

22. Mount the section with sufficient mounting media and cover with a cover slip.            Air-dry in a well-ventilated area (e.g. fume hood).

Thank you very much Dr Egidio

Dear Ivan

Is your antibody working well with mouse liver? where do you purchase? using santa cruz CK19, I cannot get any signal from mouse liver, but very good for human liver samples

Dear Ivan,

Could you please send me the protocol of mouse Ck19? I am working with this antibody, but I have some troubles.  I work with pancreas and have the same situation as Yan when it was very good for human pancreas tissues, but less for mouse sections. 

Thank you very much !

 Dear Huyen

we got anti-CK19 from dshb and it is working very well in mouse

http://dshb.biology.uiowa.edu/Antibody-Collections/FAQ?search=TROMA+III

Thank you so much, Yan.

By the way, could you let me know you are working with paraffin-embedded sections or cryostat sections? Actually, my anti-CK 19 works pretty well with paraffin section, but no signals for cryostat sections (fixed in PFA 4 % and frozen before cutting).

Thank again for your concern,

Best,

Huyen

Paraffin, havn't test with frozen section. but I think the 4%PFA shoud be applied after frozen tissue was sectioned