Ab24170

After 3 h of NK cell/A. fumigatus co-incubation the medium was topped up by RPMI (10% FCS) containing BFA, 5 µg/ml final concentration (BioLegend, # 420601) and primary rabbit anti LAMP1 ab, 8.3 µg/ml final concentration (Abcam, # ab24170). Coculture was continued for another 2 h. Sample fixation was performed by adding FA/RPMI (10% FCS) for 5 min at 37 °C with a final concentration of 1.2% FA. A second fixation step in 3% FA/RPMI (10% FCS) for 5 min at 37 °C was performed, followed by three washing steps in 0.1% Saponin/PBS. Sample blocking was performed in 5% BSA/PBS for 30 min at RT. Primary mouse anti perforin ab (BioLegend, clone dG9, # 308102) was diluted to 10 µg/ml in 0.1% Saponin/PBS and incubated for 1 h at RT, followed by three washing steps in 0.1% Saponin/PBS. Secondary goat anti-mouse Alexa Fluor 488 ab (Thermo Fisher, # A11017, fab fragment) was diluted to 10 µg/ml in 0.1% Saponin/PBS and incubated for 1 h at RT. After washing twice in 0.1% Saponin/PBS secondary donkey anti-rabbit ATTO 643 (Jackson ImmunoResearch, # 711-005-152, custom labeled with ATTO 643) was diluted to 5 µg/ml in 5% BSA/PBS for 1 h at RT. After washing twice, the sample was postfixed with 3.7% FA/0.25% GA in PBS, 10 min at RT.

Cells were enzymatically labeled, fixed in 2% paraformaldehyde, 7% sucrose, in PBS. Free aldehydic groups from paraformaldehyde were covered incubating with 50 mM NH₄Cl for 10 min. Buffer containing 0.25% Saponin, 5% normal goat serum, and 2% serum bovine albumine in PBS was used for permeabilization and blocking; incubation with antibodies was performed in the presence of 0.25% Saponin. The following primary antibodies were used: mouse antiearly endosome antigen 1 (anti-EEA1) 1:100 (610456, BD Biosciences, San Jose, CA, USA); rabbit antilysosomal-associated membrane protein 1 (anti-LAMP1) 1:500 (ab24170; abcam, Cambridge, UK); rabbit anti-Rab4 1:100 (sc-312, Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-Rab5 1:100 (sc-28570, Santa Cruz Biotechnology); rabbit anti-Rab7 1:100 (sc-10767, Santa Cruz Biotechnology); rabbit anti-Rab11 10 µg/ml (71-5300, Invitrogen); mouse antivesicle-associated membrane protein 2 (anti-VAMP2) 1:500 (Synaptic Systems, Gottingen, Germany); rabbit anti-VAMP3 1:200 (ThermoFisher Scientific); rabbit anti-Syntaxin-6 1:100 (Synaptic Systems); and mouse antitransferrin receptor (anti-TfR) 1:200 (Invitrogen). The secondary antibodies used were: goat anti-Mouse Alexa Fluor^® 405 conjugate 1:250 (Invitrogen); goat anti-Rabbit DyLight 405 conjugate 1:500 (ThermoFisher Scientific). Immunofluorescence microscopy was performed as described before.

The primary antibodies used were mouse anti-APC (1 : 1,000; PO80 Calbiochem-EMD Millipore, Billerica, MA, USA), mouse anti-GFAP (1 : 1,000; MAB3402, Chemicon-Millipore, Temecula, CA, USA), rabbit anti-GFAP (1 : 1,000; ZO334, Dako, Denmark), mouse anti-HA (1 : 100; MMS-101P, Covance Princeton, NJ 08540, USA), mouse anti-NeuN (1 : 500; MAB377, Chemicon), rabbit anti-Iba1 (1 : 200; 019-19741, Wako Chemicals, Richmond, VA, USA), rabbit anti-Lamp-1 (1 : 200; AB 24170, Abcam Cambridge Science Park, Cambridge, UK), chicken anti-GFP (1 : 1,000; AB-13970, Abcam), rabbit anti-GFP (1 : 1000; A-11122 Molecular probes; Carlsbad, CA, USA), chicken anti-GALC (CL1021AP, 1 : 2,000; a gift of Dr C.W. Lee), rabbit anti-cleaved Caspase-3 (1 : 200, Cell Signalling, Danvers, MA, USA), rat anti-CD68 (1 : 100, MCA1957, AbD Serotec, Kidlington, Oxford, UK) and anti-CD3 (molecular complex; 1 : 80, BD Biosciences Pharmigen).

Western blot analysis was performed as previously described [6 ]. Regular Western blot analysis was performed using LV lysates that were prepared using RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protein inhibitor mixture (Sigma-Aldrich)). In addition, Western blot analysis of protein aggregation was performed using LV detergent-soluble and -insoluble lysates. Briefly, detergent-soluble fractions of LV tissues were obtained using a 2% Triton X-100 lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 2% Triton X-100, and a protein inhibitor mixture (Sigma-Aldrich)). The insoluble fractions were further solubilized using the 2% Triton X-100 lysis buffer supplemented with 1% SDS. Primary antibodies included anti-LC3 (L7543, Sigma-Aldrich), anti-P62 (ab91526, abcam), anti-NQO1 (sc-376023, Santa Cruz Biotechnology, Inc.), Anti-ATG5 (A2859, Sigma-Aldrich), anti-ATG7 (A2856, Sigma-Aldrich), anti-LAMP1 (ab24170, Abcam), anticathepsin D (2284, CST), and anti-Ub (sc-8017, Santa Cruz Biotechnology, Inc.). Ubiquitinated proteins with molecular weights from the top to 35 kDa on the membranes of immunoblots were quantified