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Comparison of MCFO IHC and chemical tag labeling methods.

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posted on 2018-08-15, 17:29 authored by Geoffrey W. Meissner, Jonathan B. Grimm, Rebecca M. Johnston, Ben Sutcliffe, Julian Ng, Gregory S. X. E. Jefferis, Sebastian Cachero, Luke D. Lavis, Oz Malkesman

All samples show the Drosophila left optic lobe imaged at 63X. Each image is independently scaled for optimal intensity. Arrowheads indicate bleed-through into Cy2/AF488 channel. (A). MCFO pure IHC: Split GAL4 SS00313 was crossed to 57C10-Flp2 in attp18;; pJFRC201-10XUAS>STOP>myr::smGFP-HA in VK00005, pJFRC240-10XUAS>STOP>myr::smGFP-V5-THS-10XUAS>STOP>myr::smGFP-FLAG in su(Hw)attP1, and was labeled with nc82 mouse anti-Brp/Alexa Fluor 488 anti-mouse, rat anti-FLAG/Alexa Fluor 647 anti-rat, rabbit anti-HA/Alexa Fluor 594 anti-rabbit, and DyLight 550 mouse anti-V5 over a period of 7 days. (B). MCFO hybrid IHC: SS00313 was crossed to 57C10-Flp2 in attp18; brp-SNAP; pJFRC201-10XUAS>STOP>myr::smGFP-HA in VK00005, pJFRC240-10XUAS>STOP>myr::smGFP-V5-THS-10XUAS>STOP>myr::smGFP-FLAG in su(Hw)attP1 and labeled for 15 minutes with Cy2 SNAP-tag ligand, followed by rat anti-FLAG/Alexa Fluor 647 anti-rat, rabbit anti-HA/Alexa Fluor 594 anti-rabbit, and DyLight 550 mouse anti-V5 over a period of 6 days. (C). MCFO ATTO 647N pure IHC: As in (A) but with ATTO 647N instead of Alexa Fluor 647. (D). MCFO ATTO 647N hybrid IHC: As in (B) but with ATTO 647N instead of Alexa Fluor 647.

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