Hi folks...........

Hope everyone enjoyed the holidays and had great time with family and friends.....May we all c more peace and love on earth in the coming days and weeks..........

I presented my problem in last discussions about having high background in the whole mount preparation of around 1 mm thick tissue of foetus of rat. There were some good suggestions like:

1. Reducing the con of triton-X from Kathleen Spencer and Pam Marcum. I have done that from 1% to 0.5 and then 0.1%, but no change in results.

2. Reduce the fixation time in Zamboni from overnight to few hrs (6 hrs)....done...no result.

3. Young Kwun...suggested me to look into the work of Furnees JB from Australia, which i also did. I have some help from that, but as their tech is diff and has also used other fixatives and antibodies, hence is not really very helpful.

4. Andrew Gray from Australia, who is also doing some work in same lines...suggested to switch the antibodies...which will be my last option, but still i am scared about the end result....

During the quest of solution, in literature survey, I have found few investigators, who have used different counter stains for the quenching of high background in IH, but that all work is in sections rather than in whole mount preparations. My query from the group is:

1. Has someone used counter staining at any stage for whole mount preparation?
2. What dyes has been used and in what conc (the details of work)
3. Does these staining will affect the signals and hence the final results?
4. Or is there any other way to het rid of this "high background" in my whole mount workOO

Thanks very much for ur time, patience and also for the help.

Cheers

Parkash Mandhan

Fellow, Peads Surg

Christchurch

New Zealand