Hybond lfp pvdf transfer membranes

For WB, 6 mothers with litters and 6 pup-deprived control rats were used. The alpha-crystallin B chain (Cryab) protein showed the highest fold change among the altered proteins in the POA. Therefore, we selected this protein for validation by Western blot. Proteins were separated with Tricine-SDS-polyacrylamide gel electrophoresis using 4% stacking and 15% resolving polyacrylamide gels then transferred to Hybond-LFP PVDF transfer membranes (GE Healthcare). The membranes were blocked with 5% BSA in Tris-buffered saline, 0.1% Tween 20 (TBS-T). The blots were incubated with goat anti-Cryab primary antibody (sc-22,391, Santa Cruz Biotechnology) at 1: 1000 dilution. Subsequently, the membranes were washed for 4 × 5 min in TBS-T followed by incubation with ECL Plex CyDye conjugated anti-goat IgG secondary antibody at 1: 2500 dilution (GE Healthcare). After the washing steps, the bands were visualized using a Typhoon TRIO + scanner. The ImageQuant TL software was used for the quantification of the fluorescence intensities. The densitometry data of protein band intensities were analyzed with ImageJ software (NIH, Bethesda). Every protein band intensity was normalized to the average band intensity of the pup-deprived control group. Differences between the two samples were statistically analyzed using independent Student’s t-tests.