Ki-67 in rat liver tissue

[alinef]

Hi, i'm new in this forum but i think it's very useful.

I am trying to stain paraffin liver sections to characterize proliferating cells in the rat liver using IHC. My problem consists in the fact that primary antibody binds non-specifically to the hepatic cells nucleus resulting in a diffuse staining of the sample. I have tried:
·Peroxidase  blocking: 3% H2O2 in methanol, 15 min
·Antigen retrieval: I have already proved two protocols: 10 mM citrate pH 6.0 microwave as well as 1mM EDTA pH 8.0 95°C.
·normal serum blocking ( horse serum PK-6200 Vector, Vectastain ABC Kit)
·biotin blocking: Vector, Avidin/Biotin Blocking Kit, sp-2001
·Primary antibody: mouse anti-human Ki-67 antigen 1:50, 1:100 and 1:500 dilutions
·Secondary Ab: Universal PK-6200 (horse anti-mouse/rabbit, Vector, Vectastain ABC Kit).
· Liquid DAB Susbtrate-Chromogen System (DAKO, code n.  K3466) 3 or 5 min

Moreover, I would like to emphasize that the negative control (no primary antibody) is really negative and that I am sure that the antibody cross-reacts with rat Ki-67 since the proliferating cells were properly stained in the positive control tissue (rat oral mucous). However it could be noticed stained nucleus in the negative control tissue (rat cerebellum) as well. Could you help me?

Thanks,
Aline

[richard03]

Can you explain the followings:

Moreover, I would like to emphasize that the negative control (no primary antibody) is really negative.

However it could be noticed stained nucleus in the negative control tissue (rat cerebellum) as well. Could you help me?

Thanks,
Aline

[alinef]

Hi,
I would like to mean that when the normal serum substituted the primary antibody, the nuclei of rat liver cells were not stained.
However, the cerebellum, which does no express the Ki –67 antigens (because this it is recommended as a negative control tissue) also shows stained nuclei.
I hope I have been clearer now.
Thank you
Aline

[richard03]

Cerebellum is generally recommended as a negative control tissue for Ki67, but it may not be absolutely accurate. Some small percentage of cells may still be stained positively. Check this article:

http://fens2004.neurosciences.asso.fr/posters/R5/A145_18.html

[alinef]

Thanks for the article!
But, my bigger problem is related to the fact that all nuclei from the liver rat sections are staining after the Ki-67 IHC, including those of liver from normal rats. I am asking myself if this could be a problem with the primary antibody, since is anti-human and produced in mouse.
I would like to use an rabbit anti-rat but I have noticed that only mouse anti-rat MIB5 is available.

[ImmunoNYC]

Is your anti-mouse secondary absolutely adsorbed against rat. Can you show pictures of your negative controls, I know you say they are negative but I wonder if your anti-mouse secondary is cross reacting with your rat tissue?

On the other hand, I strongly suggest you use anti-rat Ki67 antibody specifically called MIB5 available from DAKO. You mentioned you didn't want to use this and I cannot understand why ... it works like a charm for us!

Finally are you generally interested in proliferation status or are you fixed on using Ki67? There are other options for specific phases of the cell cycles which I would be glad to tell you about if you are interested.