Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and microinjection into the ARCN or PVN.
In normal rats, leptin (RD Systems, Minneapolis, MN) was microinjected (5, 25, and 100 ng in 50 nL) into the ARCN or PVN (n = 8 rats/group).
In normal rats, NMDA receptor antagonist AP5 (16 pmol in 50 nL) was microinjected into the ARCN or PVN, 10 minutes prior to microinjection of leptin (100 ng in 50 nL; n = 8 rats/group).
In a separate group of control and T2D rats, leptin receptor siRNA was microinjected into the ARCN or PVN.
Frozen serial coronal sections (100 [micro]m/section) of the ARCN (15 sections) and PVN (6 sections) were cut with a cryostat according to a stereotaxic atlas and bilaterally punched with an 18-gauge needle using the Palkovits and Brownstein technique .
The total protein concentrations in the punched ARCN and PVN samples were measured with a bicinchoninic acid assay kit (Pierce, Rockford, IL).
He disclosed that a new substantive provost will be put in place by ARCN
, noting that application had been submitted by some interested applicant but said 'the lecturers should not be seen as being sponsored by the applicants, it is going to be to their disadvantage.
The Executive Secretary of ARCN
Mr Saidu Madagwa who was represented by Mr Yarama Ndirpaya said 'As the coordinating agency for the over 15 agricultural research institutes in Nigeria, we have identified modern biotechnology as one scientific tool whose potentials can help improve crop and animal production and we have done this with all sense of responsibility, bearing in mind both national and international protocols that guide the deployment of genetic modification.'