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As per our talk over the phone, here are the images of the double staining attached.
Tissue collection: The tissues used were rat uterus. When the rat was alive she was perfused with PBS followed by 4% PFA. Tissues collected and stored in the 4% PFA for couple of day and the dehydrated with 20% sucrose solution. The dehydrated tissues crayo-frozen and stored at -80.
Tissue preparation: The frozen tissues were sliced with 10 micro meter thick and fixed on a glass slide by 4% PFA for 10 mins. The washed with PBS (3 times). Antigen retrieved in a microwave for 10 mins then washed with ice cold PBS (2 time). The slided were blocked by 5% NGS and 3% FCS. The primary antibodies in 0.1% trinton in PBS with 1% FCS and 1% NGS. Incubated overnight at 40 C (For confirming the axact dilutions needed we tried 1:100,1:200 and 1:400 and for double staing we used 1:400).
On day two the secondary antibodies were added for ER alpha (Ab32063) FitC goat anti rabbit antibody (1:250) and for ER beta Cy3 goat anti mouse antibody (1:250) was used. Incubated for 90 mins and washed with PBS and then added was Hoescht dye (1:1000). Then washed again with PBS and finally with DM water and mounted with mowiol. Images were captured at 20X and also viewed under 40X oil emmersion (No difference).
Images attached are all for double staining. Wherein I have split the images with different fluorecence and also a double fluoroscence imaged.
My question are,
1. As Estrogen receptors belong to the nuclear receptors and it should stain nucleus largely but both the anti bodies does not show any staining into the nucleus.
2. Also, I see that in my blank slides, I see artifacts of fluorescence which is expected to at the region where I expect protein expression, which is one of the difficult situation to analyse my results. Do you have any suggestion for secondary antibodies?
Awaiting for your reply
Asked on Oct 12 2012
Thank you for sharing that information with me and sorry for the delay in getting back to you. This makes understanding the problems you have encountered easier.
In answer to your questions:
1. The ER alpha and beta would be expected to be located in the nucleus however, it is difficult to tell if you are seeing any specific nuclear staining considering the background staining your are seeing with the secondary antitbodies alone.
2. I would agree that the background you are seeing is making it difficult to interpret the results. I think I'd like to collect a little more information from you in order to be able to advise how to improve the results.
If you could let me know which secondary antibodies you are using (the supplier and catalogue number) this would be helpful. Have these secondary antibodies been used successfully with any other primary antibodies?
Have you performed any staining of any other targets in the tissue?
Which antigen retrieval buffer was used?
How long was the blocking performed for (and at what temperature)?
Was any permiabilization performed? If so, how?
What wash steps were employed between each antibody incubation?
With this information I will hopefully be able to advise as to what may be worth optimising and hopefully reduce the background staining.
I look forward to receiving your reply.
Answered on Oct 12 2012