Non-Abcam antibody was used: Donkey anti rabbit Alexa 555 (Life Technologies) Host species: Donkey Clonality: Polyclonal Conjugation: Alexa Fluor® 555
Thank you for your review of this previously untested application, IHC of PFA-fixed, frozen tissue, and for the comparison of results from two batches. For consistent staining after the lengthy fixation (2 weeks in 4% PFA before cryoprotection and freezing, according to the reviewer), an antigen retrieval step before staining may help. For heat-mediated retrieval, a temperature lower than the standard 95-100C may be necessary to minimize damage to fragile tissue. The following reference discusses optimization of an antigen retrieval protocol. J Neurosci Methods. 2000 Dec 15;104(1):27-34.
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Submitted Jul 14 2015
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