The first experiment used 10 WT and 10 FGF21 KO mice for analyzing testicular and hepatic expression of FGF21 mRNA under fas ting and low fasting conditions. For negative control, TdT was omitted from the reaction mixture. Under microscope apoptotic cells would show a brown nuclear mark because the TUNEL positive and were quantitatively counted manually. From each of the three sections at least from each testis we randomly selected 30 seminiferous tubules cross sections which were selected in a same sample to move each fall Ibrutinib 936563-96-1 without repetitive counting in a blinded manner, i. e., the examiner was unaware of the information of slides. At least 3 parts were counted from each testis, and at least 5 animals were counted in each class. The apoptotic cells were counted from spermatogonia, key spermatocytes, and secondary spermatocytes, although not spermatid and spermatozoa since whole cells of the former may be easily iden tified for the quantification. Effects were offered as TUNEL good cells per 103 cells. We also calculated the index that was the percentage of primarily round seminiferous tubules with more than three TUNEL positive cells. Thirty fields from each of the three sections at the least were measured for Ribonucleic acid (RNA) each of the five testes in each group. Western blots were done as described in our previous studies. Shortly, testicular tissues were homogenized in RIPA lysis buffer for obtaining the protein by centrifuging at 12,000 rpm at 4 C for 15 min. The testicular protein concentration was calculated. The protein sample was diluted in running buffer and heated at 95 C for 5 min, separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis at 12-0 V, and then transferred to a nitrocellulose mem brane. Membranes ubiquitin lysine were washed briefly in Tris buffered saline containing 0. Hands down the Tween 2-0 and plugged with blocking buffer for 1 h, and incubated over night at 4 C with these antibodies: anti cleaved caspase 8, anti Bax, anti Bcl 2, anti cleaved caspase 3, anti apoptosis inducing factor, and anti glucose regulated protein 78, anti cleaved caspase 1-2, anti activating transcription factor 4, and anti C/EBP homologous protein and anti actin. The membranes were incubated with the horseradish peroxidase conjugated secondary antibody for 1 h at room temperature, after-the unbound antibodies were removed. Blots were visualized utilizing an improved chemi luminescence detec tion system. All experiments were performed in triplicate and repeated at least three times. Quantitative densitometry was performed on the bands with a computer-based rating system, as employed in previous studies. Total RNA was extracted from testicular tissues using Trizol reagent.