Lane 1 : Anti-Adipose Triglyceride Lipase antibody [EPR19650] (ab207799) at 1/1000 dilution (2% Bovine Serum Albumin)
Lane 2 : Anti-Adipose Triglyceride Lipase antibody [EPR19650] (ab207799) at 1/1000 dilution (3% Milk)

All lanes : Human adipose normal tissue lysate - total protein (ab28980)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 55 kDa


Exposure time: 8 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin (lane 1) and 3% Milk (lane 2) before being incubated with ab207799 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized 3T3-L1 (mouse embryonic fibroblast cell line) undifferentiated and differentiated cells labeling Adipose Triglyceride Lipase with ab207799 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing positive staining on 3T3-L1 cells differentiated for 6 days. The level of expression in 3T3/L1 can be induced by differentiation treatment according to the literature (PMID 19297333).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded rat brown adipose tissue labeling Adipose Triglyceride Lipase with ab207799 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on rat brown adipose tissue is observed (PMID: 15550674). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat white adipose tissue labeling Adipose Triglyceride Lipase with ab207799 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on rat white adipose tissue is observed (PMID: 15550674). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse brown adipose tissue labeling Adipose Triglyceride Lipase with ab207799 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on mouse brown adipose tissue is observed (PMID: 15550674). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse white adipose tissue labeling Adipose Triglyceride Lipase with ab207799 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on mouse white adipose tissue is observed (PMID: 15550674). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500 dilution.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Adipose Triglyceride Lipase was immunoprecipitated from 0.35 mg of 3T3-L1 (mouse embryonic fibroblast cell line) differentiated for 6 days whole cell lysate with ab207799 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab207799 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: 3T3-L1 differentiated for 6 days whole cell lysate 10 µg (Input).

Lane 2: ab207799 IP in 3T3-L1 differentiated for 6 days whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207799 in 3T3-L1 differentiated for 6 days whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.

All lanes : Anti-Adipose Triglyceride Lipase antibody [EPR19650] (ab207799) at 1/500 dilution

All lanes :

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Exposure time: 1 second

IHC image of Adipose Triglyceride Lipase staining in a formalin-fixed, paraffin-embedded human adipose tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval (EDTA based pH 9.0 solution, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab207799, 1µg/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. As a negative control (inset), an identical assay was performed without adding the 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.

All lanes : Anti-Adipose Triglyceride Lipase antibody [EPR19650] (ab207799) at 1/1000 dilution (3% Milk)

Lane 1 : Adult Mouse Adipose Tissue Lysate
Lane 2 : Adult Rat Adipose Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 55 kDa


Exposure time: 5 seconds

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab207799 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.