Anti-CD36 antibody

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD36 antibody (AB124515)

IHC image of ab124515 staining in mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab124515 ,1µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

• WB

Unknown

Western blot - Anti-CD36 antibody (AB124515)

CD36 contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted. 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 5% Bovine Serum Albumin before being incubated with ab124515 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-CD36 antibody (ab124515) at 1 µg/mL

Lane 1:

Heart (Mouse) Tissue Lysate at 10 µg

Lane 2:

P7 Adipose (Mouse) Tissue Lysate at 10 µg

Lane 3:

Brown Adipose (Mouse) Tissue Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 53 kDa

Observed band size: 150 kDa,300 kDa,88 kDa

true

Exposure time: 4min

• WB

Lab

Western blot - Anti-CD36 antibody (AB124515)

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

All lanes:

Western blot - Anti-CD36 antibody (ab124515) at 1/5000 dilution

Lane 1:

Mouse platelet tissue lysate at 20 µg

Lane 2:

Mouse adipose tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 53 kDa

Observed band size: 75 kDa

false

Exposure time: 90s

• WB

CiteAb

Western blot - Anti-CD36 antibody (AB124515)

CD36 Western Blotting using Anti-CD36 antibody, ab124515. Publication image from Fernández-Hernando, C. et al., 2016, Nat Commun, PubMed : 27460411.

ANGPTL4 deficiency promotes macrophage foam cell formation and apoptosis.(a) Representative pictures from WT and Angptl4−/− mouse peritoneal macrophages incubated with or without Ac- LDL (120 μg ml−1) for 24 h and stained with BODIPY 493/503 (1 μg ml−1) and DAPI (Green and blue, respectively). Scale bar, 5 μm. Quantification of the mean average intensity is in the right panel. (b) Total cholesterol content in peritoneal macrophages isolated from WT and Angptl4−/− mice incubated with or without Ac-LDL (120 μg ml−1) for 24 h. (c) Flow cytometry analysis of DiI-Ox-LDL binding in peritoneal macrophages incubated with DiI-Ox-LDL (30 μg cholesterol per ml) for 30 min at 4 °C. At the end of the incubation period, cells were washed and incubated in RPMI 10% FBS media for 15 min at 37 °C to allow the internalization. (d) Flow cytometry analysis of DiI-Ox-LDL uptake in peritoneal macrophages incubated with DiI-Ox-LDL (30 μg cholesterol per ml) for 2 h at 37 °C. The results are expressed in terms of specific MFI after subtracting auto-fluorescence of cells incubated in the absence of DiI-Ox-LDL. (e) Cholesterol efflux to apolipoprotein A1 (ApoA1) in peritoneal macrophages isolated from WT and Angptl4−/− mice stimulated with or without T0901317 (T090). (f) Western blot analysis of indicated proteins in peritoneal macrophages from WT and Angptl4−/− mice incubated with or without Ac-LDL (120 μg ml−1) for 24 h. (g) Western blot analysis (representative of three blots) of ABCA1 expression in WT and Angptl4−/− peritoneal macrophages incubated with Ac-LDL for 24 h. Surface ABCA1 was isolated using biotinylation followed by incubation with neutravidin. HSP90 is used as loading control (f and g). Full scans of westerns blots are provided in Supplementary Fig. 8. (h) Representative confocal images of mouse peritoneal macrophages from WT and Angptl4−/− mice incubated with Ac-LDL for 24 h and stained with cholera toxin B (CTxB), ABCA1 and DAPI. Quantification of co-localization of CTxB and ABCA1 is on the right panel. Scale bar, 10 μm. (i) Representative images of WT and Angptl4−/− macrophages cultured on coverslips and treated with or without Ac-LDL (120 μg ml−1) in combination with ACAT inhibitor (58035) for 24 h to induce lipid-loading-induced apoptosis (scale bars, 200 μm). Apoptosis was detected using Annexin-V staining. Right panel shows the quantification of percentage of apoptotic cells from four random fields from each cover slip. All data represent the mean±s.e.m. from at least three experiments in duplicate; *P < 0.05 compared with WT macrophages by unpaired t-test. MFI, median intensity of fluorescence.

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