Dna polymerase 1 large klenow fragment

The xenograft tumors harvested after sacrifice were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin, then serially sectioned (4 µm) thickness, and subjected to histopathological analysis. Tissue sections were stained with hematoxylin and eosin, TUNEL stain (DNA Polymerase 1 Large (Klenow) Fragment from Promega; dATP, dCTP, dGTP from Promega, Cat # M2201; Bio-11-dUTP from Cedarlane) (Wijsman et al., 1993 (link)), Ki-67 antibody (Novus Biologicals, Cat # NB110–90592, RRID:AB_1217069) and BrdU antibody (Abcam, Cat# ab6326, RRID:AB_305426) following the manufacturer’s instructions. All slides were counterstained with hematoxylin and assessed under light microscopy. For IHC analysis – TUNEL expression levels were semi-quantified using immunoreactive scores (IRS range 0–12). Score definitions: 0 (score 0–3), 1+ (score 4–6), 2+ (score 7–9), 3+ (score 10–12). For Ki-67 and BrdU percent positivity, 10 fields were randomly chosen, and the average percentage of positive cells determined. All slides were assessed by a pathologist. Slides were scanned on the Aperio Scanscope AT2 Whole Slide Scanner using a 20x objective.

The northern blot was performed as described [26 (link)]. In brief, total RNA extracted from mouse tissues, including muscle, brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT) and spleen at 8 weeks of age were separated by PAGE (7M urea) on 6% polyacrylamide gels and transferred to a nylon membrane (N⁺; Amersham). AK079912 probes were labeled with α-32p-cytidine triphosphate (CTP) using DNA polymerase 1 Large (Klenow) Fragment (Promega, U1100). RNA blots were hybridized in ULTRAhyb (Ambion) at 68 °C overnight, washed twice (5min) with 2 × saline sodium citrate (SSC)/0.1% SDS wash buffer at 68 °C, followed by stringent washes (2 × 30min) with 0.1 × SSC/0.1% SDS wash buffer at 68 °C. RNA blots were then exposed to X-ray film at −80 °C.