Anti-ALDH1L1+ALDH1L2 抗体 [EPR25443-54] (ab300509)

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: Lane 1: 6 seconds; lane 2: 37 seconds; lane 3: 15 seconds; Lane 4: 37 seconds; lanes 5-6: 8 seconds; lane 7: 59 seconds.

Bands below 100 kDa may due to degradation (PMID: 29979702).

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time:  3 seconds. 

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labelling ALDH1L1 and ALDH1L2 with ab300509 at 1/5000 (0.107 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on astrocytes of mouse cerebrum is observed. The section was incubated with ab300509 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins is used. 

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labelling ALDH1L1 and ALDH1L2 with ab300509 at 1/5000 (0.107 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on mouse liver is observed. The section was incubated with ab300509 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins is used. 

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labelling ALDH1L1 and ALDH1L2 with ab300509 at 1/500 (1.072 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on astrocytes of rat cerebrum is observed  (PMID: 18171944) . The section was incubated with ab300509 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins is used. 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling ALDH1L1 and ALDH1L2 with ab300509 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in HepG2 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1:200 dilution (2.5 μg/mL). 

Secondary antibody only control: PBS was used instead of primary antibody followed by preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (2 μg/mL). 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse primary astrocyte cell labeling ALDH1L1 and ALDH1L2 with ab300509 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (2 μg/mL) (green). Confocal image showing cytoplasmic staining in mouse primary astrocyte. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. The nuclear counter stain is DAPI (blue). 

Astrocyte cell was counterstained with: Anti-Glial Fibrillary Acidic Protein (GFAP) mouse monoclonal antibody at 1/1000 dilution (2 μg/mL). 

The negative controls are as follows:
-ve control 1: ab300509 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution (2 μg/mL). 

-ve control 2: Anti-GFAP mouse monoclonal antibody at 1/1000 dilution, followed by ab150081 1:1000 (2 μg/mL). 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized rat primary neuron/glia cell labeling ALDH1L1 and ALDH1L2 with ab300509 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (2 μg/mL) (green). Confocal image showing cytoplasmic staining in rat primary astrocyte. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. The nuclear counter stain is DAPI (blue). 

Astrocyte cell was counterstained with: Anti-Glial Fibrillary Acidic Protein (GFAP) mouse monoclonal antibody at 1/1000 dilution (2 μg/mL). 

The negative controls are as follows:
-ve control 1: ab300509 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution (2 μg/mL). 

-ve control 2: Anti-GFAP mouse monoclonal antibody at 1/1000 dilution, followed by ab150081 1:1000 (2 μg/mL).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling  ALDH1L1 and ALDH1L2 with ab300509 at 1:100 (5.36 μg/mL) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000  dilution (2 µg/mL) (Green). Positive staining on mouse cerebrum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: PBS was used instead of ab300509 followed by preabsorbed secondary antibody (ab150081) Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000  dilution (2 µg/mL). 

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was used. 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum tissue labeling ALDH1L1 and ALDH1L2 with ab300509 at 1:100 (5.36 μg/mL) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000  dilution (2 µg/mL) (Green). Positive staining on rat cerebrum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: PBS was used instead of ab300509 followed by preabsorbed secondary antibody (ab150081) Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000  dilution (2 µg/mL). 

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 + 0.05% Tween-20) was used.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methnol permeabilized mouse primary neuron cells labelling ALDH1L1 and ALDH1L2 with ab300509 1:50 dilution (1μg)/ Right compared with a Rabbit monoclonal IgG (ab172730) (Left). Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

ALDH1L1 and ALDH1L2 was imunoprecipitated from mouse brain tissue lysate 10 μg with ab300509 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab300509 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/50000 dilution.

Lane 1: Mouse brain tissue lysate 10 μg (Input)

Lane 2: Ab300509 IP in mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300509 in human liver tissue lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 32 seconds.

ALDH1L1 and ALDH1L2 was imunoprecipitated from human liver tissue lysate (10 μg) with ab300509 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab300509 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/50000 dilution.

Lane 1: Human liver tissue lysate 10 μg (Input)

Lane 2: ab300509 IP in human liver tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300509 in human liver tissue lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.

Dot blot analysis of ALDH1L1 and ALDH1L2 using ab300509 at 1/1000 dilution followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution. 

Lane1: His-tagged recombinant mouse ALDH1L1 protein

Lane 2: His-tagged recombinant mouse ALDH1L2 protein 

Blocking and diluting buffer and concentration: 5% NFDM/TBST. 

Exposure time: 48 seconds.