Anti-galectin 9/Gal-9 antibody [EPR22214] - Low endotoxin, Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-galectin 9/Gal-9 antibody [EPR22214] - Low endotoxin, Azide free (AB246797)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney) cells labeling galectin 9/Gal-9 with ab227046 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing no staining in HEK-293T cells

Negative control : HEK-293T (PMID : 11698107). The nuclear counterstain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
Secondary antibody only control : Used PBS instead of the primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227046).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-galectin 9/Gal-9 antibody [EPR22214] - Low endotoxin, Azide free (AB246797)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling galectin 9/Gal-9 with ab227046 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear and cytoplasmic staining on Kupffer cells of human liver (PMID : 20209097, PMID : 18202194) is observed. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227046).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-galectin 9/Gal-9 antibody [EPR22214] - Low endotoxin, Azide free (AB246797)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling galectin 9/Gal-9 with ab227046 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear and cytoplasmic staining on epithelial cells of human colon (PMID : 18202194) is observed. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227046).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-galectin 9/Gal-9 antibody [EPR22214] - Low endotoxin, Azide free (AB246797)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized PANC-1 (human pancreatic epithelial cancinoma cell line) (Left) / U937 (human histiocytic lymphoma cell line) (Right) cell line labeling galectin 9/Gal-9 with ab227046 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative Control - PANC-1 cells.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227046).

• WB

Lab

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] - Low endotoxin, Azide free (AB246797)

Lanes 1 - 2 : Merged signal (red and green). Green - ab227046 observed at 35 kDa. Red - loading control Mouse anti Histone H3 observed at 18 kDa.

ab227046 was shown to react with galectin 9/Gal-9 in wild-type THP-1 cells in Western blot with loss of signal observed in LGALS9 knockout cell line ab269505 (knockout cell lysate ab269667). Wild-type THP-1 and LGALS9 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab227046 and Mouse anti Histone H3 overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

LGALS9 knockout THP-1 cell lysate at 20 µg

Predicted band size: 40 kDa

Observed band size: 35 kDa

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