Immunostaining of LC3 (green) in the arcuate hypothalamic nucleus of lean and obese mice (A; 8 weeks of a HFD or B; 16 weeks of a HFD). DAPI (blue) was used for nuclear staining.

The brain was excised after the mice were decapitated. Each SNC was fixed in 4% paraformaldehyde and each hypothalamus was processed for paraffin embedding and sectioned into 5.0 μm sections. Samples were incubated with primary antibodies overnight and with secondary antibodies conjugated to FITC or rhodamine for 2 hours (sc2777and sc2092, respectively; Santa Cruz Biotechnology, Santa Cruz, CA). The DAPI stain was used for nuclear staining while the Leica FW 4500 B microscope captured the images. Hypothalamic areas were observed according to the landmarks in the mouse brain atlas. Analysis and documentation of the results were performed using Leica Application Suite V3.6 (Switzerland).

Western Blot shows lysates of HeLa (human epithelial cell line from cervix adenocarcinoma) cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquinine for 18 hours. PVDF membrane was probed with 0.5 ug/mL ab48394 followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody. A specific band was detected for LC3B at approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.

HeLa (human epithelial cell line from cervix adenocarcinoma) cells (wild type, left; LC3B knockout HeLa, right)  stained for LC3B using ab48394 (red) at 0.3 μg/ml in ICC/IF. Primary antibody was incubated for 3 hours at room temperature, followed by NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody. Counterstained with DAPI (blue).

LC3 was detected in immersion fixed Cloroquine treated Hela cells (left) but was not detected in LC3 knockout Hela cells (right).

LC3B immunofluorescence in primary follicles of PD 13 ovaries. The red dots represent LC3b and DAPI (blue) indicated cell nuclei.

For the immunofluorescence analysis, the ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm slices. After antigen retrieval, the slides were blocked with goat serum and incubated with primary antibody (rabbit anti-LC3B at 1:200) overnight at 4°C. Alexa Fluor 594 (Invitrogen) was used as the secondary antibody in immunofluorescence assays.

 

Formalin-fixed, paraffin-embedded mouse brain tissue stained for LC3B using ab48394 at 1/200 dilution in immunohistochemical analysis. The specific signal of LC3 was detected using HRP-conjugated secondary antibody with DAB reagent, and nuclei of cells were counterstained using hematoxylin. This LC3 antibody generated a low to moderate levels of cytoplasmic staining in the glial cells. The neurons depicted a moderate to strong staining for LC3 in their cytoplasm.

Western blot shows lysates of mouse NIH/3T3 (mouse embryo fibroblast cell line) and rat PC-12 (rat adrenal gland pheochromocytoma cell line) cell lines untreated (-) or treated (+) with Chloroquine. PVDF membrane was probed with 0.5 ug/mL rabbit anti-LC3B polyclonal Antibody (ab48394), followed by 1:2000 dilution of goat anti-rabbit IgG secondary antibody.

ab48394 staining LC3B in a Rat hepatocyte by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.2% Triton X-100 in PBS and blocked with 1% Donkey serum in 0.1% PBST for 60 minutes at 21°C. Samples were incubated with primary antibody (1/50 in PBS + 1% BSA) for 3 hours at 22°C. An Alexa Fluor^® 394-conjugated Donkey anti-rabbit IgG polyclonal was used as the secondary antibody at a dilution of 1/200.

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All lanes : Anti-LC3B antibody - Autophagosome Marker (ab48394) at 1/2000 dilution

Lane 1 : Rat whole tissue lysate - Normal liver
Lane 2 : Rat whole tissue lysate - liver treated with AEE788 at 50 mg/kg 3 times a week for 1 week
Lane 3 : Rat whole tissue lysate - liver treated with RAD at 2.5 mg/kg daily for 1 week

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Developed using the ECL technique.

Performed under non-reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

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ab48394 staining LC3B (LC3-II) in HeLa cells treated with calmidazolium chloride (ab120658), by ICC/IF. Increase of LC3B (LC3-II) expression correlates with increased concentration of calmidazolium chloride, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120658 (calmidazolium chloride) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab48394 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.