Rat anti lamp 1

CNN and isomaltitol were produced by Hayashibara Co. Ltd (Okayama, Japan). D-(+)-mannose, D-(+)-glucosamine, hydrochloride theophylline, ammonium chloride (NH₄Cl), and L-DOPA (3-(3,4-dihydroxyphenyl)-L-alanine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LY294002 was obtained from Calbiochem (Darmstadt, Germany). α-melanocyte-stimulating hormone (α-MSH) was purchased from Sigma Aldrich (St. Louis, MO). Kojic acid (5-hydroxy -2-(hydroxymethyl)-4H-pyran-4-one) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Rabbit anti-tyrosinase, rabbit anti-TRP1, rabbit anti-TRP-2, and rat anti-LAMP-1 antibodies were purchased from Santa Cruz (Dallas, Texas). Rabbit anti-Pmel17(gp100) antibody was purchased from Abcam (Cambridge, UK). Mouse anti-Pmel17(HMB45) antibody and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Dako (Glostrup, Denmark). Mouse anti-MITF antibody was purchased from Exalpha Biologicals (Shirley, MA). Mouse anti-actin antibody was purchased from EMD Millipore (Temecula, CA). Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies were purchased from Life Technologies (Carlsbad, CA). Leupeptin and pepstatin were obtained from Peptide Institute, Inc. (Osaka, Japan). Complete EDTA-free Protease Inhibitor Cocktail was purchased from Roche Diagnostics (Basel, Switzerland).

Human islets were prepared from beating heart donors with appropriate ethical permission and consents as described previously (54 (link)). Islets were dissociated into single cells and, after fixation in 4% (w/v) paraformaldehyde, were treated with antibodies as below (55 (link)). Rabbit anti-TPC2 antibody (1:150) was revealed with Alexa 568-conjugated secondary antibody (1:1500, Invitrogen, Paisley, UK). Guinea pig anti-insulin (1:300, DAKO, Ely, UK), goat anti-EEA1 (1:150, Santa Cruz Biotechnology, Santa Cruz, CA), and rat anti-LAMP-1 (1:150, Santa Cruz Biotechnology) were revealed with Alexa 488 secondary antibodies (1:1500, Invitrogen). Murine MIN6 clonal β cells (56 (link)) were transfected with plasmid encoding TPC2-mCherry using Lipofectamine 2000, and 48 h later were fixed, stained, and imaged as above. Images were captured using a Zeiss Axiovert 200 M spinning disc confocal imaging system (×40 oil immersion objective corrected for chromatic aberration; Hamamatsu ImageEM 9100-13 back-illuminated EM-CCD camera) with illumination (491 and 568 nm) provided by solid state lasers (Crystal Laser, NV) using a laser merge module (Spectral Applied Physics, Ontario, Canada) (57 (link)).

At DIV 7, cultured primary neurons in 24 wells were washed once with 1X PBS, then fixed in 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 10 min. After three times washing with 1X PBS, cells were blocked with 10% normal donkey or goat serum in 1 X PBS for 30 min at RT followed by three times washing in 1 X PBS. Thereafter, cells were incubated with primary antibodies diluted in 1 X PBS containing 1% normal donkey or goat serum for 2–3 hr at RT. three times washing with 1 X PBS, incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (1:500, Invitrogen) in 1 X PBS containing 1% normal donkey or goat serum for 1 hr at RT. Washed with 1 X PBS for three times, then counterstained the slides with DAPI (1:2000, Sigma) and mounted by using Vectashield (Vector) after rinsing. Primary antibodies used in this study were rabbit anti-APP (1:100, Synaptic Systems, 127 003), mouse anti-rab5 (1:100, Synaptic Systems, 108011), mouse anti-rab11a (1:20, Santa Cruz, sc-166523), mouse anti- Golgin-97 (1:100, Invitrogen, A-21270), rat anti-Lamp1 (1:20, Santa Cruz, sc-19992). After staining, images were obtained by using confocal microscope (Olympus FV-1200 or Leica SP8). The percentage of APP or APP-ΔCRD co-localizing with rab5, rab11, Golgin-97 and Lamp1 was calculated using JACOP (Bolte and Cordelières, 2006 (link)).