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2 protocols using alexa fluor 647 a21236

1

Immunofluorescence Staining of Cellular Proteins

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Following antibodies were used: anti-GAPDH (HRP-60004, Proteintech), anti-6∗His (ab18184, Abcam), anti-GFP (11814460001, Roche), anti-Myc (2278S, Cell Signaling Technology), anti-ACBD3 (H00064746-B01P, Abnova), anti-P-PKA-substrate(9621S, Cell Signaling Technology), anti-Golgin97 (13192, Cell Signaling Technology), anti-mCherry (ab167453, Abcam), anti-PKA-Cα (#4782, Cell Signaling Technology), anti-PKA-RIIα (612242, BD), anti-γ-COP (sc-393615, Santa Cruz), anti-GM130 (610822, BD Bioscience), anti-SNAP tag (P9310S, NEB), anti-Clathrin HC (ab21679, Abcam), anti-ARF1 (NB300-505, Novus), anti-β-Actin (3700S, Cell Signaling), and anti-α-Tubulin (3873S, Cell Signaling). Anti-Rabbit Alexa Fluor 488 (A21441), Alexa Fluor 568 (A10042), Alexa Fluor 647 (A21245), and anti-Mouse Alexa Fluor 488 (A21200), Alexa Fluor 568 (A10037), and Alexa Fluor 647 (A21236) for immunofluorescence staining were obtained from ThermoFisher. D/D Solubilizer (635054) was purchased from Clontech. Forskolin (F6886) was purchased from Sigma.
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2

Immunohistochemistry Protocol for Fluorescence Imaging

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Immunohistochemistry was performed as described previously (12 (link)). Fluorescence images were acquired using Zeiss LSM 800 Confocal Microscope and processed with Fiji ImageJ imaging software.
The primary antibodies used were as follows: chicken anti-GFP (1:1,000) (ab13970; Abcam), rabbit anti-HA (1:1,000) (3724S; Cell Signaling Technologies), rabbit anti-DsRed (1:200) (632496; Takara), and mouse anti-chaoptin (1:20) (24B10; Developmental Studies Hybridoma Bank). Rat anti-dFezf (1:50) was a gift from Dr. Claude Desplan (New York University, New York, NY).
The secondary antibodies used were as follows: Goat anti-chicken immune globulin (Ig) G (H + L) Alexa Fluor 488 (A-11039), goat anti-rabbit IgG (H + L) Alexa Fluor 568 (A-11011), goat anti-mouse IgG (H + L) Alexa Fluor 647 (A-21236), and goat anti-rat IgG (H + L) Alexa Fluor 568 (A-11077) were purchased from Thermo Fischer Scientific and used at a dilution of 1:500.
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