Rabbit anti lamp1 pab

To image the localization of LAMP1 in HAP1, H1-ΔV33, and H1-ΔV33A-fV33, cells the cells were seeded onto coverslips one day prior to staining. Cells were fixed in 4% formaldehyde in PBS for 15 min at RT, washed with PBS, and subsequently permeabilized in PBS containing 0.1% Triton-X-100 for 10 min. Cells were incubated with antibody against LAMP1 (rabbit anti-LAMP1 pAb, 1∶100 dilution; Abcam) in 3% BSA in PBS followed by incubation with secondary antibodies coupled to AF488, AF-568 phalloidin, and DAPI (all Life Technologies). The samples were analyzed using a confocal laser-scanning microscope (Leica SPE-II).

The following antibodies were utilized for western blot and immunofluorescence studies. Mouse anti-PI3,4P2 mAb and mouse anti-PI3,4,5P3 mAb conjugated with FITC from Echelon Bioscience (Salt Lake City, UT). Rabbit anti-EEA1 mAb from Cell Signaling Technology (Danvers, MA). Goat anti-rabbit Ig-HRP conjugate, goat anti-mouse Ig-HRP conjugate, and goat anti-mouse pAb conjugated with Alexa Fluro 488 from Invitrogen (Carlsbad, CA). Rabbit anti-LAMP1 pAb, and rabbit anti-Rab5 pAb from Abcam (Cambridge, England). Mouse anti-Rab7 mAb from Sigma Aldrich (St. Louis, MO). For phagocytosis and phagosome maturation assays, pHrodo™ Red E. coli BioParticles™ Conjugate and DQ™-BSA Red (used in 96 well plate assay) and DQ™-BSA -Green (used for live cell assay) was purchased from Invitrogen.

Live fibroblasts derived from a Gaucher III patient (Gaslini Institute cell line F0971986) grown in 96-well black glass bottomed microplates (In Vitro Scientific, Mountain View CA) were incubated with 5uM 5(6)-(2-dimethylaminoethyl)carboxamido-2',7'-dichlorofluorescein diacetate (5) for 1 hour at 37°C/5% CO₂. Staining media was then removed from the cells and 100uL of fixative (4% formaldehyde/1% glutaraldehyde in PBS) added, followed by incubation at room temperature for 15 minutes with shaking. Cells were then washed 3 times with PBS and then permeabilized with 100uL of 0.2% Triton-X in PBS for 5 minutes at room temperature. Cells were washed 3 times in blocking buffer (1% BSA in PBS) and then incubated overnight at 4°C in blocking buffer. Primary antibody, rabbit anti-LAMP1 pAb (Abcam, Cambridge, MA) was then applied at 1.6ug/mL in blocking buffer and the cells incubated at room temperature for 1 hour. Cells were washed 3 times in blocking buffer. Secondary antibody, Alexa Fluor^® 555 conjugated Goat Anti-Rabbit (Abcam, Cambridge MA) was added at a 1:500 dilution in blocking buffer and incubated for 1 hour at room temperature protected from light. Cells were washed 3 times in PBS with Hoechst 33342 (Sigma Aldrich) added to the second wash. Images were then captured on a Zeiss Axio Observer A1 inverted microscope fitted with a 40X lens and DAPI, FITC and TAMRA filter sets.