Anti-LC3B 抗体 [EPR18709] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192890).

ab192890 staining LC3B in paraffin embedded human astrocytoma tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.  Samples were incubated with primary antibody at 1/1000 dilution for 30 mins at room temperature. Ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used as the secondary antibody. Hematoxylin was used as a counterstain. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

ab192890 staining LC3B in HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 labeled with DAPI (shown in blue).

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

Alexa Fluor^® 488 (ab225383) and Alexa Fluor^® 647 (ab225382) conjugated versions are available for this clone.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192890).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

Clone EPR18709 (ab221794) has been successfully conjugated by Abcam. This image was generated using Anti-LC3B antibody [EPR18709] - Autophagosome Marker (Alexa Fluor® 488). Please refer to ab225382 for protocol details.

ab225382 staining LC3B in HeLa chloroquine-treated (50µM, 24 hours) cells. 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 ab225453 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

Clone EPR18709 (ab221794) has been successfully conjugated by Abcam. This image was generated using Anti-LC3B antibody [EPR18709] - Autophagosome Marker (Alexa Fluor® 647). Please refer to ab225383 for protocol details.

ab225383 staining LC3B in wild-type HAP1 cells and knockout cells, untreated and chloroquine-treated (50μM, 24 hours). The cells were fixed with 100% methanol (5min), 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 with ab225383 at 0.5μg/ml (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192890).

Immunohistochemical analysis of formalin fixed paraffin embedded human cortex labelling LC3B with ab192890 at a dilution of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5) . ab192890 anti LC3B antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

IHC image of LC3B staining in a section of formalin-fixed paraffin-embedded normal human cerebral cortex* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab221794, 0.1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

This ICC data was generated using the same anti-LC3B antibody clone, EPR18709, in a different buffer formulation (cat# ab192890).
ab192890 staining LC3B in HAP1 cells (wildtype and MAP1LC3B knockout) +/- 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 1 μg/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 labeled with DAPI (shown in blue).

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

• IP

Supplier Data

Immunoprecipitation - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

LC3B was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab192890 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab192890 at 1/1000 dilution. Veriblot for IP (HRP) (ab131366), was used for detection at 1/1000 dilution.
Lane 1 : HeLa whole cell lysate 10μg (Input).
Lane 2 : ab192890 IP in Jurkat whole cell lysate.
Lane 3 : Rabbit IgG,monoclonal [EPR25A] -Isotype Control (ab172730) instead of ab192890 in HeLa whole cell cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 30 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192890).

All lanes:

Immunoprecipitation - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (<a href='/products/primary-antibodies/lc3b-antibody-epr18709-autophagosome-marker-ab192890'>ab192890</a>)

Predicted band size: 15 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192890). False colour image of Western blot : Anti-LC3B antibody staining at 1 ug/ml, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab63817 was shown to bind specifically to LC3B. A band was observed at 16/14 kDa (yellow arrows) in treated wild-type HepG2 cell lysates with no signal observed at this size in MAP1LC3B knockout cell line ab277828 (knockout cell lysate ab283796). To generate this image, wild-type and MAP1LC3B knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (<a href='/products/primary-antibodies/lc3b-antibody-epr18709-autophagosome-marker-ab192890'>ab192890</a>) at 1/1000 dilution

Lane 1:

Wild-type HepG2 untreated control cell lysate at 20 µg

Lane 2:

Wild-type HepG2 Treated Chloroquine (50 uM, 16 h) cell lysate at 20 µg

Lane 2:

Western blot - Human MAP1LC3B knockout Hep G2 cell line (ab277828)

Lane 2:

Western blot - Human MAP1LC3B knockout Hep G2 cell lysate (ab283796)

Lane 3:

MAP1LC3B knockout HepG2 untreated control cell lysate at 20 µg

Lane 4:

MAP1LC3B knockout HepG2 Treated Chloroquine (50 uM, 16 h) cell lysate at 20 µg

Lane 5:

U-87 MG cell lysate at 20 µg

Lane 6:

PC-12 cell lysate at 20 µg

Predicted band size: 15 kDa

Observed band size: 14 kDa,16 kDa

false

• WB

Lab

Western blot - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

This data was developed using ab192890, the same antibody clone in a different buffer formulation. Different batches of ab192890 were tested onU-87 MG (Human glioblastoma-astrocytoma epithelial cell line) lysate at 0.9 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 14,16 kDa.

All lanes:

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (<a href='/products/primary-antibodies/lc3b-antibody-epr18709-autophagosome-marker-ab192890'>ab192890</a>)

Predicted band size: 15 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody [EPR18709] - BSA and Azide free (AB221794)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192890). Tissue Microarrays stained for Anti-LC3B antibody [EPR18709] - Autophagosome Marker using ab192890 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins. The section was incubated with ab192890 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.