ab192890 staining LC3B in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab192890 at 1μg/ml and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in pseudocolor red) followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab108338, at 1/100, staining ATG9A in paraffin-embedded Human colon tissue by Immunohistochemistry.

Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling Beclin 1 with ab207612 at 1/200 dilution. Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton-X100 in PBS. Samples were incubated with primary antibody (1/200 in PBS) for 16 hours at 22°C. ab150081, a goat anti-rabbit IgG H + L  (Alexa Fluor® 488) was used as the secondary antibody at 1/200 dilution.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: ATG4B knockout HAP1 cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab154843 observed at 47 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab154843 was shown to specifically react with ATG4B when ATG4B knockout samples were used. Wild-type and ATG4B knockout samples were subjected to SDS-PAGE. ab154843 and ab18058 (loading control to Vinculin) were diluted at 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling ATG16L1 with ab187671 at 1/100 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: APG5L/ATG5 knockout HAP1 cell lysate (20 µg) 
Lane 3: Raji cell lysate (20 µg)
Lane 4: Jeg-3 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab108327 observed at 52 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab108327 was shown to specifically react with APG5L/ATG5 when APG5L/ATG5 knockout samples were used. Wild-type and APG5L/ATG5 knockout samples were subjected to SDS-PAGE. ab108327 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.