[Histonet] IF staining on FFPE tissue

Margaryan, Naira NMargaryan <@t> childrensmemorial.org
Wed Dec 23 12:55:19 CST 2009

Dear Histonetters,

Hopefully you are enjoying a great Holidays with your friends and family!

I have been asked to perform . I got positive and negative absolutely identical with beautiful nuclear DAPI and very red RBC. What to do or what to change in my protocol to avoid RBC and background? Here is my protocol:
1.Deparaffinization  (xyline- 60min, 100%Eth- 15min, 95%- 5min, 70%- 5min, H2O)
2. Antigen Retrieval ph6 in Citrate buffer (same I use for IHC)
3. Wash H2O and TBST
4. Protein block- 10minhttps://webmail.childrensmemorial.org/owa/?ae=PreFormAction&t=IPM.Note&a=Reply&id=RgAAAADBaOhrx0l9S5XoN3P8OXzpBwDbYZXQd%2bZ%2fQIud7XQY1kjHAAAKzOB%2bAADBupMEDGuaSo2Eh4%2bT%2fsNqAAOO6ORIAAAJ#
5. Ab, Rb anti-human - 60-90 min (same I use for IHC)
6. Wash TBST -5-10min
7. secondary Donkey anti-Rb 594 red - 60min
8. Wash TBST -5-10min, H2O -5min
9. DAPI - 10min
10. Wash H2O -5-10min
11. Gelvatol coverslip

Am I using wrong secondary? Is there any specific secondary or protocol for IF staining for FFPE  to avoid background? I just used same AR and primary Ab's dilution like i use for IHC with nice results.

Any suggestions are appreciated, especially in these Holidays days from working histo- people:)

Thanks and have a warm and nice Holidays,

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