Sqstm1 p62

One week after HI injury, microdissected brain tissue of the affected cortex, striatum, and corpus callosum from the injured (ipsilateral) and uninjured (contralateral) hemispheres was collected either as a single wedge of infarcted tissue, or as microdissected regions. The tissue was homogenized and then sonicated in lysis buffer. 30μg of denature protein was loaded into a 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA), and 5 μL of Amersham ECL Rainbow Marker was loaded as a molecular weight standard (GE Life Sciences, Pittsburgh, PA). Proteins were transferred onto nitrocellulose and incubated with primary antibody: LC3 (rabbit polyclonal, Cell Signaling, 1:1000), SQSTM1/p62 (guinea pig polyclonal, Progen 1:1000), β-Tubulin (mouse monoclonal, Santa Cruz, 1:000). Membranes probed for LC3 and β-Tubulin were washed with 0.01% TBS-Triton X, incubated in HRP-conjugated secondary antibody, washed, and bands visualized using Western Lightning chemiluminescence reagent (PerkinElmer, Wellesley, MA). Membranes probed for p62 were likewise washed with 0.01% TBS-Triton X and incubated in IRDye 680LT secondary antibody (LI-COR; Lincoln, NE). Imaging for LC3 and β-Tubulin was performed using a BioRad ChemiDoc Imaging System combined with Image Lab software (Hercules, CA). Imaging for p62 was performed using a LI-COR Odyssey Imaging System. Quantification was performed using ImageJ software.