The Dispute Over Ruthless Ku-0059436-Systems

Tissues were cut in pieces (10�C15?mg), washed again 3x in PBS and incubated in Dulbecco's modified Eagle's medium (DMEM/F-12, Life technologies) Ku-0059436 in vitro containing 1% essential fatty acid-free bovine serum albumin (Sigma) for 4?h at 37?��C in a humidified incubator containing 5% CO2. Thereafter, the supernatants were removed and analyzed for FGF21 (R&D Systems). BAT and iWAT tissue pieces were washed in PBS, weighted, snap frozen in liquid nitrogen and stored at??80?��C for protein detection. 2.5. Ex?vivo analysis of FGF21 secretion in soleus and EDL muscle Ex?vivo analysis of FGF21 secreted from EDL and soleus muscles was measured as described before [11]. 2.6. Histology Fat tissue specimens were fixed in 4% paraformaldehyde (Roth chemicals) for 24?h and embedded in low melting paraffin (Paraplast Plus?, Sigma Aldrich) for histological examination. Four ��m-thick sections were cut using a rotary microtome (HSM55, Microm). Sections were mounted on superfrost glass slides (Menzel glass), dehydrated in increasing ethanol series and stained with hematoxylin and eosin (H&E) (Merck). Bright field images were obtained with the Keyence Microscope BZ-9000. 2.7. Statistics Statistical analyses were performed using Stat Graph Prism (6.0) (Graphpad). All data are reported as mean?��?SEM. After testing for normal distribution of the data and equal variances within the data sets, a Student's t-test (unpaired, two-tailed) was used to determine differences between the genotypes under different temperatures, whereby asterisks indicate the degree of statistical significance which was assumed P? letters indicate significant different at P?