Rhodamine conjugated secondary antibody

Sulforhodamine B, BrdU, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich. Antibodies against β-actin, α-tubulin, γ-tubulin , and BrdU (Sigma-Aldrich) and pericentrin (Covance) were obtained from the indicated sources. Cep70 antibody was generated as described previously15 (link). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Rhodamine-conjugated secondary antibody was from Jackson ImmunoResearch Laboratories. GFP-Cep70 plasmid, Cep70 siRNAs, and luciferase control siRNA were described previously16 (link).

Primary antibodies for immunoblotting were from Cell Signaling Technology (Beverly, MA, USA) (Table 2). Anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were from Bio-Rad (Hercules, CA, USA). Goat anti-desmin antibody, used for cytochemistry, was from DakoCytomation (Glostrup, Denmark). Rhodamine-conjugated secondary antibody was from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Rats were sacrificed by intracardial perfusion of 0.1 mM PBS (pH 7.4) followed by 4% paraformaldehyde. Brains were immersed in 4% paraformaldehyde at 4 °C for 12 h for fixation and then immersed in a 30% sucrose solution for dehydration. Seven-micrometer-thick coronal frozen sections were collected. Immunofluorescence staining was performed as previously described^{37 (link)}. The primary antibodies were anti-Beclin-1 (ab16998, 1:200, Abcam), anti-NeuN (MAB377, 1;500, Millipore), anti-GFAP (ab53554, 1:500; Abcam), Iba-1 (ab107159, 1:500; Abcam), with a rhodamine-conjugated secondary antibody (1:200, Jackson Immuno Research) and a fluorescein isothiocyanate-labeled secondary antibody (1:200, Jackson Immuno Research). The sections were rinsed, stained with DAPI (1 µg/ml, Roche Inc, Basel, Switzerland), rinsed again and mounted with glycerol. Fluorescence microscope (Olympus) was used to analyze the labeling.
For Fluoro-Jade C staining, specimens were dried for 30 min, rehydrated, and then incubated in 0.06% potassium permanganate solution for 10 min. After a water rinse, the slices were stained with 0.0001% Fluoro-Jade C solution for 5 min. Finally, the slices were dried again, cleared in xylene, and cover-slipped with mounting medium.

Immunocytochemistry assays were performed on cells seeded in 8-well plates (Daigger Scientific, Vernon Hills, IL, USA) as reported previously. The cells were fixed with 3.7% PFA in PBS at 4 °C, washed, permeabilized with DAKO target retrieval solution (Dako Cytomation, Carpinteria, CA, USA), and blocked with 2% albumin at room temperature. Then, the cells were incubated with monoclonal antibody [MMP2, MMP9, pGSK3β (Ser⁹), GSK3β, and pβ-catenin (Ser⁴⁵), pβ-catenin (Ser^33/37), β-catenin, pJNK (Thr¹⁸³ and Tyr185), JNK, pERK (Tyr²⁰⁴), ERK] diluted at 1:100 (Santa Cruz Biotechnology) for 1 h at room temperature and developed either with FITC or rhodamine-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at a 1:200 dilution. The slides were mounted in DAPI solution (Vector Laboratories, Inc. Burlingame, CA, USA) and observed under a Fluoview FV1000 inverted laser scanning confocal microscope (Olympus).

The Flag-tagged wild-type AVPR2 and AVPR2-∆V279 plasmids were generated by subcloning each target gene’s full-length cDNA into the BamHI XhoI sites of a pCMV-3Tag-1A vector (Yao-Hong Biotechnology, New Taipei City, Taiwan). Human embryonic kidney 293T (HEK293T) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Thermo Fisher Scientific, Waltham, MA, USA). 293T cells (5 × 10⁴) were co-transfected with 2 μg of wild-type or mutant AVPR2 and the CherryPicker Cell Capture plasmids (Takara Bio, Shiga, Japan) using a calcium phosphate transfection procedure [19 (link)]. The Flag-tagged epitope-specific mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) was used for immune-staining, followed by phosphate buffered saline (PBS) washing. Then, Flag-tagged wild-type or mutant AVPR2 was visualized using a rhodamine-conjugated secondary antibody (Jackson ImmunoResearch, Philadelphia, PA, USA). ProLong^® Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Life Technologies, Carlsbad, CA, USA) was used for nucleus staining. Fluorescence confocal microscopy was performed using a confocal microscope set, CARV II™ Confocal Imager, equipped with an inverted Olympus IX71S8F3 optical microscope.

The antibodies used for this study were AMYLASE (A8273; Sigma-Aldrich), Atg5 (PM050, MBL, Woburn, MA), phos-TFEB (ABE1971; Millipore, Burlington, MA), TFEB (A303-673A; Bethyl Laboratories, Montgomery, TX), CK19 (TROMA-III, Developmental Studies Hybridoma Bank, Iowa City, IA), GAPDH (2118; Cell Signaling Technology, Danvers, MA), p62 (H00008878-M01; Abnova, Taipei, Taiwan), phos-4EBP1 (9451; Cell Signaling Technology), 4EBP1 (9452; Cell Signaling Technology), phos-MAPK (9101; Cell Signaling Technology), MAPK (9102; Cell Signaling Technology), phos-S6-ribosomal protein (4858; Cell Signaling Technology), S6-ribosomal protein (2217; Cell Signaling Technology), GFP (sc-9996; Santa Cruz Biotechnology, Dallas, TX), LAMIN A/C (2032; Cell Signaling Technology), LAMP1 (1D4B and H4A3; Developmental Studies Hybridoma Bank, Iowa City, IA), LAMP2 (ABL-93 and H4A4; Developmental Studies Hybridoma Bank), and PGC1α (PAB12061; Abnova). The TAP antibody was generated by Thomas Kolodecik from Yale University.⁴⁵ (link) Antibodies for VATP6V1a and VATP6V1b2 are gifts from Dr. Dennis Brown from Harvard Medical School. The Anti-LC3 antibody was generated as previously described.⁴³ (link) HRP-conjugated, FITC-conjugated and Rhodamine-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA).