Fig. 2. Histologic, K8/K18/K19/K20 protein and mRNA analysis in caerulein-induced pancreatitis. (A) Mice were injected with caerulein (50 µg/kg, ip) seven times at hourly intervals, and euthanized at different time points (1-240 hours), after which pancreata were fixed then stained with hematoxylin and eosin. All time-points are given as hour after the first injection, and control mice were euthanized 48 hours after vehicle-alone administration (7 saline injections). a=control; b=6 hours after injection of caerulein 6x hourly; c=48 hours after injection of caeruelin 7x hourly; and d=240 hours after injection of caeruelin 7x hourly. (B) After caerulein injections (see Materials and Methods) pancreata were collected at the indicated time-points (two mice (1, 2) per time-point) then homogenized. Protein homogenates were separated by SDS-PAGE (20 µg/lane) then blotted using antibodies to the indicated antigens. Control mice `C' were euthanized 48 hours after seven saline injections. (C) HSE or pancreatic tissue homogenates were prepared from mice injected with caerulein or saline (48 hours, two mice/condition). The amount of keratin isolated by HSE was normalized to the content of the detergent-soluble supernatant protein that was generated during the first step of the HSE procedure (see Materials and Methods), then analyzed by SDS-PAGE and densitometric scanning (to quantify fold-increase in keratins upon caerulein exposure). (D) Total RNA was isolated from pancreata 48 hours after caerulein or saline injection (two mice (M1, M2) per condition) followed by RT-PCR amplification of actin or the indicated keratin cDNAs. (E) Total pancreas RNA was isolated 48 hours after saline (2 mice) or caerulein (3 mice) treatment, or from the small intestine (SI, used as a control for K20 expression) of Balb/c mice (M1). The RNA was reverse transcribed using K20 primers (upper panel), followed by nested PCR using internal K20 primers (middle panel). HSE from the indicated tissues were also obtained and blotted using K20-specific Ab (lower panel). No sample was loaded in lane 6 in order to separate the small intestinal control from the remaining samples.