H 160

Immuno-EM was basically performed by the previously described method (47 (link)). After perfusion fixation with 2% (vol/vol) paraformaldehyde and 2% (vol/vol) glutaraldehyde (GA) in 0.1 M phosphate buffer (PB), brain tissues were further postfixed for 1 h at 4 °C. Then cerebellum tissues of 40 μm in thickness were prepared with a vibratome (Leica). After pretreatment with 0.05% Triton-X100 in PBS for 20 min at 4 °C, the tissues were incubated in 5% normal goat serum for 30 min at 4 °C. The tissues were incubated with anti-Shh (1:10; Santa Cruz, H-160) in PBS overnight at 4 °C, followed by incubation with anti-rabbit IgG labeled with 1.4-nm gold particles (1:50) in PBS overnight at 4 °C. After washing, the labeled tissues were postfixed in 2% GA in 0.1 M PB at 4 °C overnight. After washing again, the tissues were gradually dehydrated with a stepped series of ethanol and eventually embedded in epoxy resin (Quetol-812; Nisshin EM). An electron microscope at 80 kV (JEM-1400Plus; JEOL) was used to observe ultrathin sections of 80 nm in thickness. Positive particles per one view (4 μm × 4 μm) in the EGL and somas of PCs from three mice of each strain were determined.

Dieosin glutathione disulfide, Di-Eo-GSSG, was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased upon reduction of its disulfide bond51 (link). Reductase activity of purified enzymes was monitored in 96-well fluorescence microtiter plates. Recombinant ERp5, ERp72, PDI and ERp57 were assayed at the concentrations indicated in the absence or presence of anti-PDI antibody directed at an epitope in the b′ domain (H-160; Santa Cruz) or at an N-terminal epitope in PDI (PH4B; Aviva) (3 μM). Di-Eo-GSSG (150 nM) was added to enzyme in the presence of 5 μM DTT and the increase in fluorescence due to release of eosin-glutathione for ERp5, ERp72, ER57 and PDI was determined by excitation at 520 nm and emission at 545 nm in a Synergy 4 plate reader,Winooski VT). The reduction of 150 nM di-E-GSSG by 5 μM DTT alone served as a negative control.

To localize FcεR1α and Nhe1 to vascular macrophages, we performed immunofluorescent triple staining on acetone fixed frozen sections from mouse atherosclerotic lesions and normal aortas. Slides were blocked with PBS containing 5% BSA for 1 h at room temperature and then incubated with the following antibodies: goat polyclonal FcεR1α antibody (G-14) (1:50, Cat# sc-33484, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal NHE-1 antibody (H-160) (1:200, Cat# sc-28758, Santa Cruz Biotechnology), and rat anti-mouse/human Mac-2 (1:100, Cat# CL8942LE, Cedarlane, Burlington, ON, Canada). The secondary antibodies were Alexa Fluor 488 (1:300, Cat# A-11006, Thermo Fisher Scientific), Alexa Fluor 555 (1:300, Cat# A-21432, Thermo Fisher Scientific), or Alexa Fluor 647 (1:500, Cat# A-21244, Thermo Fisher Scientific). The nuclei were counterstained with DAPI (1:10, Cat# R37606, Thermo Fisher Scientific). Slides were mounted using Fluorescence Mounting Medium (Cat# S3023, DAKO) and images were collected under an Olympus FluoView™ FV1000 confocal microscope.

We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R&D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) (Chen, 2006 (link)), rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

After perfusion fixation with Bouin's solution, cerebelli from 0.5 to 21-day-old mice were immersed in the same solution overnight or for 1 day. Sagittal sections of 6 μm in thickness were used for morphological analyses. Hematoxylin-eosin (H&E) staining was performed with paraffin sections. Immunostaining with antibodies against c-Ret (1:150; Immuno Biological Laboratories), phosphorylated c-Ret Y1062 (1:50; Abcam), and Shh (1:100; Santa Cruz, H-160) diluted with Can Get Signal immunostaining solution (TOYOBO) was performed with paraffin and frozen sections (19 (link), 45 ). Immunostaining with antibodies against GABRA6 (1:1000; Chemicon), calbindin D28k (1:150; Chemicon), BLBP (1:200, Abcam), PAX6 (1:500, Abcam), and Ki67 (1:500, Abcam) was performed with paraffin sections. The VECTASTAIN Elite ABC kit (Vector), Envision kit/HRP (diaminobenzidine; DAB) (DAKO) with counterstaining of hematoxylin and Alexa Fluor 488-labeled donkey anti-rabbit IgG (1:1000, Invitrogen) with counterstaining of 4′, 6-diamidino-2-phenylindole (DAPI) were used. We validated the primary antibodies used in this study with no positive signals in the specimens processed under the same staining condition except for incubation without primary antibodies.