Ab10983

Western blots were performed as previously described. Briefly, total protein lysates from iWAT and BAT (180 and 30 μg) samples were separated by SDS-PAGE, electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, MA, USA) and probed with antibodies against Ucp1 (ab10983, Abcam, Cambridge, United Kingdom), and alpha-tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA). The antibodies dilution was 1:1000. For protein detection biotinylated secondary antibody conjugates, diluted (1:5000) and chemiluminescence (Thermo Fisher Scientific, Madrid, Spain) were used. Then, the membranes were exposed to radiograph film (Super RX, Fuji Medical X-Ray Film; Fujifilm, Tokyo, Japan) and developed with developer and fixing liquids (AGFA, Mortsel, Belgium) under appropriate dark room conditions. Protein levels were relatively quantitated by normalization with alpha-tubulin levels in the same lysate. The total or absolute protein levels were calculated by multiplying the specific Ucp1 protein level per mg homogenate protein with the total amount of homogenate protein as previously described25 (link).

At necropsy, liver and fat tissues from helminth-infected, non-infected were collected, frozen in Tissue Tek OCT compound (Miles, Inc., Elkhart, IN) and stored at −80 °C. Ten-μm sections were cut on a Leica CM1850 Cryostat (Leica Biosystem) and were stained with hematoxylin and eosin or anti-UCP1 antibody. For the fat tissues, the samples from brown adipose, subcutaneous and gonadal adipose tissues were prepared. Using the histopathological approach, we examined the sizes of adipocytes from the various groups of mice that were fed with HFD or CD with and without H. polygyrus infection. Both brown and white adipose tissue sections were processed and UCP1 expression of adipose tissues was determined using immunohistochemical staining with anti-UCP1 antibody (ab10983, Abcam, Cambridge, MA, US).

Liver, BAT and inguinal WAT sections were fixed with 10% formalin. Paraffin-embedded 7 μm sections were then stained with hematoxylin and eosin according to standard laboratory protocols. In WAT sections, Ucp1 staining was performed using UCP1 antibody (ab10983, Abcam) diluted to 1/500.

E-WAT from mice was perfused and fixed in 10% formalin saline, and then paraffin embedded. Paraffin-embedded sections were cut to 4 μM and the slides stained with hematoxylin and eosin. For uncoupling protein 1 (UCP1) staining, slides were deparaffinized, and antigen retrieval was carried out by heating sections to 95°C in sodium citrate buffer. Sections were blocked with goat serum before incubation with rabbit anti-UCP1 antibody (ab10983; Abcam Inc., Cambridge, United Kingdom) diluted 1:100. Following peroxidase blocking, horseradish peroxidase-conjugated goat anti-rabbit (P0448; Dako, Glostrup, Denmark) was used as the secondary antibody, and sections were incubated at 1:1000 in PBS for 1 h at room temperature. For staining, diaminobenzidine chromagen (K3468; Dako) was used according to the manufacturer’s instructions, and Mayer’s hematoxylin was used to counterstain.