At the end of study, animals were humanely euthanized using a CO2 fume chamber at a rate of 2 L/min for 3 min, then perfused by hand pressure with 60 mL of 1× phosphate-buffered saline (PBS) in a 60 mL syringe with a SURFLO winged infusion set size 23Gx¾ (Terumo, NJ). The heart, lung, liver, spleen, kidney, and spinal cord were harvested and immediately snap frozen and stored at −80 °C. The brain was microdissected into 12 regions: left and right hemispheres of cerebellum, cortex, hippocampus, striatum, olfactory bulb, and thalamus/brainstem, and immediately snap frozen and stored at −80 °C.
Organs were processed using a Bullet blender STORM bead mill homogenizer (Next Advance) as previously described [20 ]. In brief, organs were cut into small pieces and transferred into preassigned 1.5 mL locked-cap tubes containing 200–400 μL of sterilized saline solution. The tubes were placed in the Bullet blender for homogenization at speed 12 for 5 min. Tissue homogenates were transferred into new GeneMate 1.5 mL microtubes (VWR, PA) and stored at −20 °C for qPCR. The remaining tissue homogenates were centrifuged at 13,000 rpm for 15 min at 4 °C. The entire supernatant was then transferred into a new 1.5 mL microtube and stored at −20 °C to −80 °C for IDS, GAG, and protein assay.
Organs were processed using a Bullet blender STORM bead mill homogenizer (Next Advance) as previously described [20 ]. In brief, organs were cut into small pieces and transferred into preassigned 1.5 mL locked-cap tubes containing 200–400 μL of sterilized saline solution. The tubes were placed in the Bullet blender for homogenization at speed 12 for 5 min. Tissue homogenates were transferred into new GeneMate 1.5 mL microtubes (VWR, PA) and stored at −20 °C for qPCR. The remaining tissue homogenates were centrifuged at 13,000 rpm for 15 min at 4 °C. The entire supernatant was then transferred into a new 1.5 mL microtube and stored at −20 °C to −80 °C for IDS, GAG, and protein assay.