3.4. Endosome isolation by OptiPrep™ density gradient centrifugation from mouse brain tissue

Mice are anesthetized and transcardially perfused with saline (PBS) as in 3.3.3 and 3.3.4.

Collect the brain and use ½ for the subcellular fractionation (a specific brain regions can be dissected and used as well).

Quickly weight out the brain tissue and add 10x vol/brain weight homogenization buffer (e.g. for an average weight of 0.15-0.2 g use 1.5-2 ml of homogenization buffer).

Homogenize the tissue with 30-40 strokes of a Teflon-coated pestle in ice.

Centrifuge the samples at 1,000 x g for 20 min to pellet nuclei and unbroken tissue and obtain a post nuclear supernatant (PNS).

Collect the PNS and determine sample protein concentration (e.g. from one hemibrain homogenized in 1.4 ml of buffer the concentration of the PNS is about 7 mg/ml).

Adjust 5-6 mg of PNS in homogenization buffer to 25% OptiPrep™ adding an equal volume of 50% stock solution to a final volume of 2 ml. Keep an aliquot of the adjusted PNS as input (this will be mixed with Laemmli Sample Buffer followed by heat denaturation for western blot analysis).

Set up the gradient by loading the PNS in 25% OptiPrep™ at the bottom of a clear centrifuge tube and consecutively overlay with 1.5 ml of 20%, 15%, 14%, 12.5% and 10% and 5% OptiPrep™ solutions.

Load the tube on a SW 40 rotor and centrifuge overnight (18 h) at 100,000 x g at 4 °C.

After centrifugation carefully collect 22 x 0.5 ml aliquots from the top of the tube and analyze by Western Blot by loading equal volumes for each fraction collected, following resuspension in 5X Laemmli sample buffer and heat denaturation at 95 °C for 5 min.

Probe the blots with primary antibodies against different subcellular compartments (Fig. 5), such as rab5 for early endosomes (Abcam); Rab7 (Abcam) for late endosomes; Cathepsin D (made in house) for lysosomes; Tom20 (Santa Cruz Biotechnology) for mitochondria; sec61B for endoplasmic reticulum (ER) (Proteintech); p58K for cis-Golgi (Sigma); syntaxin6 for trans-Golgi network (TGN) (Cell Signaling Technology). The following enrichment is expected at given Optiprep™ %: 5-10%, late endosomes; 10-12.5%, early endosomes; 12.5-15%, TGN; 15-20%, ER; 15-25%, lysosomes; 20-25%, mitochondria; 25%, Golgi. This method has been optimized from [18].

a, Adult mouse brain homogenates were fractionated with an iodixanol step gradient and 22 fractions collected from the top. Equal volumes of each fraction (to show enrichment), along with the PNS input, are subject to Western blot analysis. Shown are the distributions of Rab5 (early endosome, EE), Rab7 (late endosome, LE), syntaxin6 (trans-golgi network, TGN), cathepsin D (lysosome, Lys), Sec61B (endoplasmic reticulum, ER), Tom20 (mitochondria, mito) and p58 (Golgi). Membrane proteins, weakly stained by Ponceau red, are enriched between 5-20 % Optiprep™).