Anti lamp1 antibody

Cells were washed twice with PBS then fixed using 2% (w/v) formaldehyde/0.2% glutaraldehyde in PBS for 20 min prior to a brief wash with PBS and incubation with ice-cooled methanol on ice for 10 min. After a brief rinse with PBS, cells were permeabilised and blocked through incubation with 5% (v/v) FCS/0.1% (v/v) tween 20 in PBS for 20 min followed by 4 × 5 min washes with PBS. Anti-LAMP1 antibody (Abcam) was incubated at a dilution of 1/200 in 0.5% (v/v) FCS in PBS overnight at 4 °C, followed by washing and incubation with FITC-conjugated anti-rabbit IgG for 30 min. The cells were then washed 4 × 5 min in PBS, incubated with DAPI to stain DNA, washed in PBS and imaged using a Nikon Spinning Disk confocal microscope.

The following primary antibodies were used; CD11a Alexa Fluor^™ 594 clone HI111 (Biolegend, San Diego, CA), rabbit polyclonal anti-Rab5, anti-Rab11A, anti-Rab7, or anti-Lamp1 antibody (Abcam, Cambridge, UK), anti-beta-actin antibody (AnaSpec, Fremont, CA) (1:1000), and anti-LtxA monoclonal antibody 107A3A3 [75 (link)] in hybridoma supernatants (1:10 dilution). The following secondary antibodies were used: goat anti-rabbit IgG Alexa Fluor^®488 (1:1000); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Fc) or (HRP)-goat anti-rabbit (Pierce, Rockford, IL) (1:10,000). Transferrin labeled with Alexa Fluor®555 was from Invitrogen (Waltham, MA, USA). Dynamin inhibitor Dynole 34-2 and its inactive control, Dynole 31-2, were purchased from SigmaAldrich (St. Louis, MO), Dynasore and Pitstop 2 (Abcam, Cambridge, UK). The inhibitors were used in the following concentrations: 10 μM Dynole 34-2; 10 μM Dynole 31-2; 10 μM Dynasore; 5 μM Pitstop 2.

After deparaffinization, rehydration, and antigen retrieval, the sections were stained with an anti-LC3B antibody (Novus Biologicals, NB100-2220, Co., United States), anti-TNF-α antibody (Proteintech, 17590-1-AP), anti-LAMP1 antibody (Abcam, ab24170), anti-Syntaxin 17 antibody (Proteintech, 17815-1-AP) and DAPI (Invitrogen, Carlsbad, United States). The stained tissues were imaged using a confocal microscope (LSM 880, Zeiss, Oberkochen, Germany).

Sixteen female SCID mice (Charles River „CB17/lcr-PrkdcSCID/lcrlcocrl”) were locally shaved and 3 × 10⁶ HUH-7 cells were injected subcutaneously into the flank of each mouse. Mice were divided into two groups and treated intraperitoneally with 0.2 mg/kg archazolid in 5% DMSO/10% solutol/PBS or equal amounts of 5% DMSO/10% solutol/PBS. Mice were treated daily. Measurement of tumors was done every 2 to 3 days with a caliper, using the formula a × b²/2. The average tumor volumes of the two groups were compared over time. IHC analysis of tumor tissue sections was performed as described previously [49 (link)] using anti-LAMP1-antibody (Abcam), filipin (Sigma Aldrich), anti-Ki67-antibody and haematoxylin (Sigma Aldrich). Animal experiments were approved by the District Government of Upper Bavaria in accordance with the German animal welfare and institutional guidelines.

Cells were seeded onto poly-l-lysine-coated glass cover-slides. For binding experiments, cells were incubated with biotinylated nanocages in DMEM 10% FBS for 1 h at 4 °C, washed in PBS, fixed in 4% paraformaldehyde, and incubated for 5 min with NaBH₄. For uptake experiments, cells were incubated with nanocages at 37 °C for different time periods, fixed with 4% paraformaldehyde, and permeabilized for 4 min with Tris/Triton (Tris HCl 0.1 M, Triton 0.1%, pH 7.7). Rabbit polyclonal anti-flotillin-1 antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) was used to detect the flotillin-1 protein. Early endosomes were visualized with polyclonal EEA1 antibody (Abcam Inc., Toronto, ON, Canada) and lysosomes with mouse monoclonal anti-LAMP-1 antibody (Abcam Inc., Toronto, ON, Canada). Donkey anti-rabbit IgG and donkey anti-mouse IgG, both Rhodamine Red-X-conjugated AffiniPure (Jackson ImmunoResearch, Cambridgeshire, UK) were used as secondary antibodies [24 (link)]. Biotinylated cages were detected by using streptavidin–FITC (Jackson ImmunoResearch, Cambridgeshire, UK). The nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA). Images were obtained with a laser confocal fluorescent microscope Olympus FV1000 at 60× magnification, and the fluorescence signal was evaluated with the IMARIS software. Co-localization events were evaluated as previously described [24 (link)].