Application
Immunocytochemistry
We have used this secondary antibody for immunocytochemistry with 2,2'-thiodiethanol (TDE) mounting media. TDE quenches the fluorescence of conventional green fluorophores (e.g., Alexafluor 488, GFP, etc....), but the original description of this mounting media (PMID: 17131355) reported an increase in the relative quantum yield of Cy3. We tried this Cy2-conjugated secondary antibody following TDE mounting of stained HEK293 cells and saw excellent signal from the green channel. We did not observe bleaching of the signal following Z-stack image acquisition.
The protocol for obtaining the image shown in the figure was a standard ICC protocol, followed by the TDE mounting procedure from the original TDE paper (PMID: 17131355), using sequential immersions in increasing concentrations of TDE. Slides were imaged on a Zeiss Observer Z1 with LED-based excitation (488 nm). Acquired images were deconvolved and the included image is a maximum intensity projection.
The protocol for obtaining the image shown in the figure was a standard ICC protocol, followed by the TDE mounting procedure from the original TDE paper (PMID: 17131355), using sequential immersions in increasing concentrations of TDE. Slides were imaged on a Zeiss Observer Z1 with LED-based excitation (488 nm). Acquired images were deconvolved and the included image is a maximum intensity projection.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Dr. Sam Nowitzki
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提交于 Jul 09 2015