Tissue sections were deparaffinized and pretreated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase Crenolanib activity. For staining with anti-p62/SQSTM1 antibody, antigens were retrieved by heating sections at 80°C in 10 mmol/L citrate buffer, pH 6.0, for 3 h prior to the hydrogen peroxide treatment. After non-specific binding was blocked with 10% normal goat serum (NGS), sections were incubated with primary antibodies at 4°C overnight.

All antibodies were diluted in 10% NGS. Sections were washed in PBS and then incubated with secondary anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (Envision+ System, DakoCytomation) at room temperature for 1 h. Reactions were visualized with 0.4 mg/mL 3,3′-diaminobenzidine (DAB) in PBS containing 0.006% H2O2 for 10 min. Nuclei were counterstained with hematoxylin. Transmission electron microscopy

was performed as previously described.[7] Gefitinib mouse Formalin-fixed specimens were dissected into 1 mm3 pieces, and were then post-fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (PB; pH 7.4) for 4 h and 1% OsO4 in PB at 4°C for 1 h. Specimens were dehydrated using a graded series of alcohols and QY-1 (Nisshin EM Co., Ltd, Tokyo, Japan), and then embedded in Quetol 812 (Nisshin EM). Ultrathin sections were cut with an LKB ultramicrotome (LKB-Produkter, Bromma, Sweden), and sections were counterstained with aqueous TI-blue (Nisshin EM) and Sato’s lead citrate.[8] Sections were examined using a 1200EX transmission electron microscope (JEOL Ltd, Tokyo, Japan). Written informed consent was obtained from the patient’s parents for the genomic analysis and for publication of the results. Genomic DNA was extracted from frozen liver and spleen using standard protocols. PCR primers were designed to amplify all the exons of NPC1 and flanking intron

regions. Direct sequencing Sinomenine of PCR products was performed using a 3130xl genetic analyzer (Applied Biosystems, Foster City, CA, USA), and sequence data were analyzed as previously described.[9] At autopsy, the spleen weighed 169 g, slightly heavier than usual. The liver weighed 1058 g and hepatomegaly was not apparent. The pancreas was hemorrhagic in the head, body and tail, indicative of acute hemorrhagic pancreatitis. The brain weight was 731 g. Gross neuropathological findings included marked atrophy of the frontal and temporal lobes bilaterally (Fig. 2a), cerebellum, brainstem and spinal cord. Coronal sections of the cerebrum exhibited marked atrophy of the deep white matter with thinning of the corpus callosum, marked atrophy of the frontal and temporal cortices and mild to moderate atrophy of the parietal and occipital cortices.