Anti lamp1 antibody

If not otherwise specified, chemicals and reagents were purchased from Sigma (Steinheim, Germany) or Roth (Karlsruhe, Germany). Pefabloc was obtained from Roche (Mannheim, Germany) and staurosporine was purchased from Enzo (Lausen, Switzerland). Ultrapure low-melting-point agarose, slice culture media, supplements and heat inactivated horse serum were obtained from Invitrogen (Darmstadt, Germany). The bicinchoninic acid assay kit was purchased from Pierce (Thermo Scientific, Rockford, USA). The ECL plus chemiluminescence kit was purchased from GE Healthcare (Buckinghamshire, UK). Monoclonal anti-PrP antibody 4H11 has been described previously (Ertmer et al., 2004 (link)). The antibody is directed against the globular domain of the protein. Anti-actin antibody was obtained from MP Biomedicals (Eschwede, Germany) and rabbit polyclonal anti-β-3-tubulin antibody was purchased from Covance HISS Diagnostics (Freiburg, Germany). Anti-calbindin D-28K antibody was purchased from Swant (Marly, Switzerland), anti-GFAP antibody was obtained from Dako Cytomation (Hamburg, Germany), anti-iba-1 antibody was purchased from Wako Pure Chemicals Industries (Osaka, Japan), and anti-lamp-1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Fluorescein-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany) or from Life Technologies (Darmstadt, Germany).

Alveolar macrophages (3 × 10⁴), isolated from wild-type and TPC2 KO mice (described in Grimm et al., 2014 (link)) were seeded on 8-well plates (Ibidi) and cultured overnight. Cells were washed once with Minimum Essential Media (MEM) supplemented with 10 mM HEPES and then treated with TPC2-A1-N or TPC2-A1-P as indicated. Ionomycin (4 µM for 10 min) was used as positive control. Following treatment, cells were incubated with an anti-LAMP1 antibody (1:200, SantaCruz) in MEM supplemented with 10 mM HEPES and 1% BSA for 20 min on ice. Cells were then fixed with 2.6% PFA (Thermo Fisher) for 20 min and incubated with Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher) for 1 hr in PBS containing 1% BSA. Nuclei were stained with DAPI. Confocal images were acquired using an LSM 880 microscope (Zeiss) with 40X magnification.