Hello,

we fixed our smears in 4% buffered formalin for 10min, then washing 5 min in physiological sodium chloride solution, then washing in distilled water and start the pretreatment.

smears can be fixed in absolute alcohol or cold acetone and then IHC can be performed.

I suggest to use poly-L lysine or silane coated slides, if not already done

Dear Bharat, Please check the specification sheet of your antibody for fixation recommendations, as well as google search for the antibody catalog number + methanol or ethanol fixation. There are antibodies to Ki67 which work with a wide range of fixations (PFA, alcohols) - please see e.g. abcam antibody spec. sheet

http://www.abcam.com/ki67-antibody-ab15580.html

When you look at the description of the method used for the first set of microphotographs (IF on HAP1 cells) - cells were fixed in 100% methanol for 5 min. That eliminates the need for antigen retrieval step. Good luck with your experiments.

Thanks a lot all of you for your support.

I suggest to use air dried, direkt smears. Fix in methanol and postfix with formalin.

HRT is necessary.