Vector universal elite abc kits

Brains from each group (n = 4) were washed in phosphate buffered saline (pH 7.4), and were subsequently processed. Brain sections from different groups of rats (n = 4 at each time point for each rat) were used for IHC using R.T.U Vectastain Universal Elite^® ABC Kit (Vector Laboratories, USA). Tissue sections were incubated with primary antibodies GSK3 (Ser9/21), GSK3β (Tyr216), total GSK3β, Cdk5, and p35/p25 at 4°C overnight. Subsequent procedures were performed according to the manufacturer’s instruction (Vector Universal elite^® ABC kits, Vector Laboratories, USA). A series of four representative images per section was collected. IHC analyses were scored according to the intensity of staining and percentage of positive cells stained with respective antibodies. The scoring was done as follows: 0, <5% (negative); 1, 5–25% (weak); 2, 25–50% (moderate); 3, 50–75% (strong), and 4, >75% (very strong) [16 (link)]. The analysis (n = 4) was done in triplicate.

Brains from each group were washed in 1X PBS, and were subsequently used in tissue processing. Each brain from rat was cut into smaller pieces using a tissue slicer and then processed using an automated tissue processor (Leica ASP 300 S Tissue Processor, Leica Microsystem, USA), embedded in paraffin wax and then sectioned using a microtome (Medite, Medizintechnik, Germany) into 4-μm thickness. Brain sections from different groups of rats (n = 2 at each time point for each rat) were used for IHC using R.T.U Vectastain Universal Elite ^® ABC Kit (Vector Laboratories). Deparaffinised brain tissue sections were heat-induced using sodium citrate buffer (10 mM sodium citrate, 0.05% tween 20, pH 6.0) and quenched with 0.3% hydrogen peroxide, blocked with 2.5% normal horse serum. Primary antibodies such as [IR-β, p-Akt (Ser 473), Akt and GLUT4] were incubated at 4°C for whole night. Subsequent procedure was done according to manufacturer’s direction (Vector Universal elite ^® ABC kits, Vector Laboratories). Finally, tissues were counterstained with Hematoxylin, dehydrated and mounted. These sections were observed using a light microscope with lens objective 40x (Olympus, USA).