[Histonet] where is my stain!

[Till, Renee]

I have done Pten on rat mammary gland for several years now with very
little problems. My only changes to the protocol have been to add the
avidin/biotin blocks and switch to a fab2 secondary, which has all but
eliminated the non-specific staining I was getting. The tissues I am
working with now, I have worked with others that come from the exact
same conditions. In a sense they are almost the controls, the normal
animals, to compare to the ones used for the cancer research. I cannot
get my pten stain to work. It is the same antibody, same protocol, same
everything that I have always used or done to the tissues.

I asked the company if they had made any changes to their antibody and
was told no. I tried another pten from another company and I get the
same results. There are only 5 or so of my 14 tissues that ever have
even the slightest hint of a stain. 2 to 3 of those come out "normal",
with staining like I would expect, with another couple only having the
most minimal staining in spots. The others are completely negative for
what I am looking for. Some of them do have a brown artifact like
staining around the structures, or in the luminal areas, but that is it.
There is absolutely no reason I can think of that these rats would not
express pten, and I can't think of anything I've done different. If it
is one of my reagents, I don't know why a couple animals, always the
same ones, would work fine, but none of the others. The original
antibody that I used I had always done at about 1:100 dilution. I could
not get even a 1:25 to work (which is when we decided to try another
antibody, which does give me some of the stain at 1:100. And not that I
put much stock in it, but 1:100 is also the company's recommended
dilution). 

I have not done any other stains on these tissues, but another tech has
used them and had no problems for her stains. 

Any ideas? Anyone an expert on pten? It's never been one of the finicky
stains, but....maybe there's something I'm not thinking of. 

 

Renee' Till, HT

Research Assistant

Arkansas Children's Nutrition Center

1212 Marshall St./N2021

Little Rock, AR 72002

Office (501)364-2785

Fax (501)364-3161

 


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