Anti-Galectin 3 抗体 [EP2775Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

ICC/IF image of unpurified ab76245 stained Panc-1 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab76245 at 10μg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor^® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43μM for 1hour at room temperature.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Galectin 3 with purified ab76245 at 1/50 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung squamous carcinoma tissue labelling Galectin 3 with unpurified ab76245.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Immunocytochemistry/Immunofluorescence analysis of HT-29 cells labelling Galectin with purified ab76245 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/500) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling Galectin 3 with purified ab76245 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Overlay histogram showing THP1 cells stained with unpurified ab76245 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% human serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76245, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labeling Galectin 3 with ab76245 at 1/100 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed secondary antibody at 1/1000 dilution. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution. The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Confocal image showing nuclear and cytoplasmic staining in RAW 264.7 cell line.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling Galectin 3 with ab76245 at 1 : 50 dilution (1μg)/ Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (Cell without incubation with primary antibody and secondary antibody (Blue)). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C6 (rat glial tumor glial cell) cells labelling Galectin 3 with ab76245 at 1 : 50 dilution (1μg)/ Red compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (Cell without incubation with primary antibody and secondary antibody (Blue)). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Immunohistochemical analysis of formaldehyde fixed mouse lung tissue sections labelling Galectin 3 with ab76245 at a dilution of 1/6000. The secondary antibody used was biotin conjugated goat anti rabbit IgG at a dilution of 1/300. Antigen retrieval was heat mediated using citric acid.

This image is courtesy of an Abreview submitted by Carl Hobbs.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C6 cells labeling Galectin 3 with ab76245 at 1/100 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed secondary antibody at 1/1000 dilution. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution. The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Confocal image showing cytoplasmic and weak nuclear staining in C6 cell line.

• WB

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Western blot - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/10000 dilution

Lane 1:

A375 cell lysate at 20 µg

Lane 2:

HeLa cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 26 kDa

Observed band size: 31 kDa

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• WB

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Western blot - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

All lanes:

Western blot - Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/10000 dilution

Lane 1:

A375 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

SW480 cell lysate at 10 µg

Lane 4:

A431 cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 26 kDa

Observed band size: 31 kDa

false

• WB

Unknown

Western blot - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/10000 dilution

Lane 1:

RAW264.7 cell lysate at 20 µg

Lane 2:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 26 kDa

Observed band size: 31 kDa

false

• WB

Supplier Data

Western blot - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

False colour image of Western blot : Anti-Galectin 3 antibody [EP2775Y] staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab76245 was shown to bind specifically to Galectin 3. A band was observed at 30 kDa in wild-type A549 cell lysates with no signal observed at this size in LGALS3 knockout cell line. To generate this image, wild-type and LGALS3 knockout A549 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-Galectin 3 antibody [EP2775Y] (ab76245) at 1/5000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

LGALS3 knockout A549 cell lysate at 20 µg

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

LNCaP cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 26 kDa

Observed band size: 30 kDa

false

• WB

Lab

Western blot - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Blocking and diluting buffer and concentration : 5% NFDM/TBST, ab129002 was used as a loading control for Vinculin.

All lanes:

Western blot - Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 3:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

SK-N-MC (human brain epithelial cell) whole cell lysate at 20 µg

Lane 6:

LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Blocking and diluting buffer : 5% NFDM/TBST.

Gal-3 expression is low in the healthy brain (PMID : 34831271, PMID : 31006066).

All lanes:

Western blot - Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 26 kDa

Observed band size: 30 kDa

false

Exposure time: 40s

• WB

Lab

Western blot - Anti-Galectin 3 antibody [EP2775Y] (AB76245)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure time : Lane 1 : 180 seconds, Lanes 2 and 3 : 15 seconds.

All lanes:

Western blot - Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 2:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa

false