Brain has the highest level of autophagy relative to other tissues. A). Brain, lung, heart, skeletal muscle, spleen, kidney and liver were collected from non-stroked 22–24 week old male C57BL/6 J mice. The tissues were assessed for the autophagy markers, LC3 I and II, and the loading control β-actin for non-muscle tissues and brain and GAPDH for muscle tissues and brain. The top subpanel in (A) is a 5-fold longer exposure than the subpanel immediately below it to highlight LC3I/II in tissues that were below the level of detection in the lower subpanel. The lower subpanel (exposed for 1 minute) shows the expression of brain LC3I/II relative to other tissues better, and with a better dynamic range, without the loss of linearity seen with the longer exposure. All subpanels in (A) are from the same blot, one tissue not used in other blots assessed in panels B &C was cropped out. A white space was left to show where the cropping occurred. B). Quantitation of non-muscle tissues normalized to β-actin and compared to brain. All quantitations were performed on film exposed for one minute, with equal antibody concentrations and incubation used on each tissue set. C). Quantitation of muscle tissues and brain normalized to GAPDH and compared to brain. D). Two saline treated and two chloroquine treated brain samples are shown as representative examples. The chloroquine treated mice show apparent accumulation of LC3II following chloroquine treatment (five hours with 60 mg/kg chloroquine) to assess autophagic flux. E). LC3II levels in brain samples from four chloroquine treated and five saline treated mice were measured. The accumulation of LC3II is noticeable, but not significantly different when quantitated against GAPDH. (* = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.0001).