RNA probes have been subsequently labeled with DIG UTP employing T7 SP6 polymerase reactions with one mg of linearized plasmid. In situ hybridization of E9. five, E14. five embryo and isolated islet sections was carried out as described in Prado et al. In short, cryostat sections have been handled with 1 mg ml proteinase K and fixed in 4% paraformaldhyde. Sections were hybridized with one mg ml of probe overnight at 70uC. High stringency washes were made use of to remove unbound probe. Sections were subsequently blocked with 10% FBS, 1% Blocking Reagent and incubated with anti digoxigenin alkaline phosphatase antibody diluted one one thousand. Slides have been washed and colour formulated making use of BM purple like a substrate. Immunohistochemistry was carried out on islet cryo sections following in situ hybridisation. Sections had been stained with guinea pig anti Insulin or guinea pig anti Glucagon. Immunohistochemistry was also carried out on paraffin sections of E14.
five mouse embryos, at the same time as E16. 5, E18. five and grownup ICR pancreata. Sections have been co stained with rabbit anti Myt3 and guinea pig anti Insulin, guinea pig anti Glucagon, guinea pig anti PP, goat anti Somatostatin or top article mouse anti Pdx1. Primary antibodies were detected employing donkey anti rabbit Alexa 488, goat anti guinea pig Alexa 546, goat anti mouse Alexa 546 or donkey anti goat Alexa 546. The Myt3 antibody was created by OpenBiosystems and was raised against the synthetic peptide RKGGIKMTPTKEEKEDSELR. describes it The serum from your terminal bleed of two rabbits was affinity purified. Mouse Upkeep, Islet Isolations and Cell Culture Mice have been maintained in accordance to the suggestions on the Canadian Council on Animal Care. All protocols have been accredited through the UBC Animal Care Committee.
Hand picked pancreatic islets had been isolated as previously described and cultured in RPMI 1640 supplemented with 10% FBS, 50U ml Penicillin Streptomycin and two mM L Glutamine at 37u in the 5% CO2 humidified incubator. mPAC cells had been maintained in Dulbeccos Modified Eagle Medium supplemented with 10% FBS, 50 U ml Penicillin Streptomycin and two mM L Glutamine at 37u in 5% CO2 humidified incubator. Islets had been cultured in 3 mM, 7 mM, 11 mM, 16. 7 mM and 33 mM glucose, or with different cytokine combinations, IL 1b and TNFa as appropriate. For cycloheximide experi ments, islets have been preincubated in 3 mM glucose for six hrs and CHX or DMSO was extra one hr prior to transferring islets to fresh 3 mM or 16. 7 mM glucose supplemented with CHX or DMSO. Database Evaluation Serial Examination of Gene Expression information had been obtained from the Mouse Atlas of Gene Expression Database. Foxa2 and Pdx1 Chromatin Immunoprecip itation sequencing data had been obtained from the Short Study Archive. Mafa and Neurod1 ChIP sequencing information were obtained in the Gene Expression Omnibus. Data have been analyzed as previously de scribed.