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[7][“S Metabolism”

We always compared mice of the same sex given that we did not investigate sex effects in this study.Randomization and blinding was conducted for treatment administration when possible, as stated below.Cerebral blood ow data were used as predened criteria to exclude animals from the study.In brief, anesthesia was induced with isourane in a mixture of O and NO and it was maintained with. A longitudinal cut was produced in the ventral middle line of the neck to expose and ligate the right common carotid artery. Next the submaxillary glands and the omohyoid and sternothyroid muscles were separated, exposing the carotid arteries.Mice received analgesia and were kept on a thermal blanket for hour after surgery.Cerebral blood ow mice with incorrect surgery or surgical complication; mice that did not show a drop in CBF greater than from basal CBF values after introducing the lament in the middle cerebral artery, as it was considered that ischemia was not successfully induced; and mice that did not show recovery in CBF of higher than of the basal value as reperfusion did not reach an adequate level.The inhibitor was mixed into AINA standard chow at ppm received the diet ad libitum for three weeks prior to induction of ischemia and the diet was maintained until the mice were killed.Treatment controls received AINA chow for the same period of time.Both diets were given in parallel in groups of animals per cage.Researchers conducting ischemia and obtaining further data were not aware of the identity of the diet groups.Ischemia was performed days after pump implantation and mice were euthanized days postischemia.Treatment controls received the same volume of the vehicle. The dose of J was decided following previous studies. Treatment was randomly assigned and was administered in a blinded fashion.The animals received a code that did not reveal the identity of the groups.Unspecic binding of antibodies was blocked by previous incubation for min with anti CDCD in FACS buffer at C.The digested tissue was ltered twice with mm and mm lters washing with HBSS. Cells were separated from myelin and debris by isotonic percoll gradient. Samples were centrifuged at xg for min without acceleration or brake.The digested tissue was ltered in mm lters washing with HBSS. ACSA cells were collected using magnetic eld columns.Cells were resuspended in FACS buffer. Fc receptors were blocked by previous incubation for min with CD CD in FACS buffer at C.Cells were cocultured for days at a: APC to T cell ratio in RPMI complete medium in the presence of recombinant murine IL. RNA was precipitated with ethanol overnight at C.Samples had RIN values. The second strand of the cDNA incorporated dUTP in place of dTTP.Library amplication was performed by PCR on selected fragments using the primer cocktail supplied in the kit.Long distance PCR was performed for was used for quantication and validation of the obtained cDNA.Briey, fragments were subjected to end repair and addition of A bases to ends, ligation of adapters and USER excision.Library amplication was performed through cycles of PCR using different indexed primers for multiplexing.

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