Cy2 conjugated goat anti mouse igg

Cells on glass coverslips were washed once with phosphate-buffered salt solution (PBS: 10 mM sodium phosphate pH 7.4, 145 mM sodium chloride, 5 mM potassium chloride) and fixed for 10 min in 4% paraformaldehyde in PBS. After washing three times with PBS, non-specific binding sites were blocked with 1% bovine serum albumin, 0.5% NP-40 in PBS for one hour. Incubation with primary antibodies was performed overnight at 4 °C in blocking solution. Primary antibodies used were rabbit anti-GM130 (dilution 1:200; cat# ab52649, Abcam, Cambridge, UK) and mouse anti-calreticulin (dilution 1:200; cat# 612136, BD Biosciences, Heidelberg, Germany). Secondary antibodies were Cy3-conjugated goat anti-rabbit IgG (dilution 1:400; cat# 111-165-144, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and Cy2-conjugated goat anti-mouse IgG (dilution 1:400; cat# 115-225-146, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Specimens were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher, Waltham, MA, USA) and viewed using an Axiovert M200 microscope (Carl Zeiss, Jena, Germany).

Primary antibodies used: Akt #9272 (1:1000), p-Akt (Ser473) # 4058 (1:1000), p-Akt (Thr308) #2965, Rab8A #6975 (1:1000), Rab10 #8127 (1:1000), AS160 #2670 (1:1000), p-AS160 (Thr642) #4288 (1:1000), IR beta #3025 (1:1000), p-IGF-IR beta (Tyr1131)/IR beta (Tyr1146) #3021 (1:1000) (all from Cell Signalling); LRRK2 (3514-1 (MJFF2) Epitomics); tubulin (ab6160, abcam); SLC2A4 (ARP43785_P050, avivasysbio, 1:100); GLUT4 (MAB1262, R&D Systems, 1:1000); Alexa Fluor 488 Mouse anti-β-Tubulin, Class III Clone TUJ1 #560338 (BD Pharmingen).
Secondary antibodies used: anti-rabbit IgG, HRP-linked #7074 (1:5000, Cell signalling); Goat anti-rat IgG, HRP-linked (1:20 000, ab6845, abcam); Rabbit anti-sheep IgG, HRP-linked (1:10 000, 31480, ThermoScientific); Goat anti-mouse IgG, HRP-linked (1:10 000, 172–1011, Bio-Rad); Cy3-conjugated Goat anti-rabbit IgG (1:350, 111-165-046, Jackson ImmunoResearch); Cy2-conjugated Goat anti-mouse IgG (1:350, 115-225-164, Jackson ImmunoResearch).

Immunofluorescence was performed as previously described^{19 (link)}. Briefly, immunofluorescence detection was conducted with antibodies against RCP (1:500, Cell Signaling Technology), E-cadherin (1:500, Santa Cruz Biotechnology), β1 integrin (1:500, Santa Cruz Biotechnology), MT1-MMP (1:500, Santa Cruz Biotechnology), and LAMP1 (1:500, Abcam, Cambridge, UK) overnight. The cells were washed with ice-cold PBS and incubated with Cy3-conjugated goat anti-rabbit IgG (1:500, Jackson ImmunoResearch, PA), Cy2-conjugated goat anti-mouse IgG (1:500, Jackson ImmunoResearch) and Cy5-conjugated goat anti-rat IgG (1:500, Jackson ImmunoResearch). The nuclei of cells were marked with 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen). The cells were observed by confocal microscopy (x600, LSM710, Carl Zeiss).