Recombinant Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (ab150423)

Fluorescence multiplex immunohistochemical analysis of the human kidney (Formalin/PFA-fixed paraffin-embedded sections).

Panel A: merged staining of anti-Hexokinase 1 (ab150423, green; Opal™690), anti-Angiotensin Converting Enzyme 1 (ab254222, gray; Opal™520) and anti-Aquaporin 2 (ab199975, red; Opal™570) on human kidney. Panel B: anti-Aquaporin 2 stained on collecting tubules. Panel C: anti-Angiotensin Converting Enzyme 1 stained on proximal tubules. Panel D: anti-Hexokinase 1 stained on distal tubules and collecting tubules. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

The section was incubated in three rounds of staining: in the order of ab150423 at 1/250 dilution (4.224 μg/ml), ab254222 at 1/4000 dilution (0.141 μg/ml) and ab199975 at 1/4000 dilution (0.152 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

Fluorescence multiplex immunohistochemical analysis of paraffin-embedded Human kidney tissue.

Panel A: Merged staining of anti-Hexokinase 1 (gray; Opal™690), anti-Angiotensin Converting Enzyme 1 (green; Opal™520) and anti-Tissue Factor (red; Opal™570) on human kidney.
Panel B: Anti-Tissue Factor stained on renal glomeruli.
Panel C: Anti-Angiotensin Converting Enzyme 1 stained on proximal tubules.
Panel D: Anti-Hexokinase 1 stained on distal tubules.

The section was incubated in three rounds of staining: in the order of ab150423, ab254222, and ab252918 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Counterstained with DAPI.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat kidney tissue sections labeling Hexokinase 1 with purified ab150423 at 1/50 dilution (4.14 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse kidney tissue sections labeling Hexokinase 1 with purified ab150423 at 1/50 dilution (4.14 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling Hexokinase 1 with purified ab150423 at 1/50 dilution (4.14 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (ab150423) at 1/1000 dilution (Purified)

Lane 1 : Human brain lysate
Lane 2 : Mouse brain lysate
Lane 3 : Rat brain lysate
Lane 4 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 5 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 102 kDa

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling Hexokinase 1 with Purified ab150423 at 1/20 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488 ,ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

 

Immunocytochemistry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Hexokinase 1 with Purified ab150423 at 1:50 dilution (4.1 µg/ml). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488,ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (ab150423) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HK1 knockout HeLa cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : HEPG2 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 102 kDa
Observed band size: 102 kDa

Lanes 1-4: Merged signal (red and green). Green - ab150423 observed at 102 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab150423 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial was shown to specifically react with Hexokinase 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267279 (knockout cell lysate ab257161) was used. Wild-type and Hexokinase 1 knockout samples were subjected to SDS-PAGE. ab150423 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 10000 dilution for 1 hour at room temperature before imaging.