Anti-Wnt1 抗体 (ab15251)

Thank you for contacting us.

It may be, as we discussed, that the PFA fixation of the the frozen tissue is the reason the immunostain is not working. I am attaching three papers that deal with antigen retrieval for IHC of frozen tissue. Two of the papers have protocols and the other is a review of antigen retrieval. Section 4.6 of the review by D'Amico is the part of the review that I think is relevant to your frozen tissue.

Since you were able to see a positive stain with the paraffin-embedded tissue without antigen retrieval, it is possible that the heat of the paraffin-embedding itself was sufficient to reverse some of the cross-linkages that were created by fixation of the tissue. That is why I am suggesting that heat-mediated retrieval of the frozen tissue sections may help develop a stain, even thought the paraffin-embedded tissue did not require a heat-mediated retrieval. I suggest trying the protocol I mentioned: Heat 10mM sodium citrate, pH 6.0, to 90C and immerse the slides in a rack in this for 30 minutes. Maintain the temperature at 90C by keeping the container with the slides in a waterbath. After 30 minutes, allow the buffer to cool for another 30 minutes, then rinse the slides in PBS, room temperature, and continue with the blocking step and the primary antibody.

You should test one slide first to see if this damages the section, before trying with many slides.

You mentioned that the tissue for the frozen samples were fixed for two hours in PFA. Was this also the length of time that the paraffin-embedded tissues were fixed, before embedding? If the tissue that was embedded in paraffin was fixed for a a longer time, then there is also the possibility that the frozen tissue samples were under-fixed.

Thank you for your enquiry. The immunogen sequence used to raise this product is highly similar to the zebra fish, exhibiting a difference of 2 out of 17aa. If you have any further questions then please get back in touch with me.