Alexa fluor 594 f ab 2 fragment of goat anti mouse igg h l

Collected cells were washed and resuspended in staining buffer (PBS 1X +1% BSA) with Fc Receptor Binding Inhibitor Antibody (Invitrogen, # 14-9161-73, Carlsbad, CA, USA) for 15 min at 4 °C. After blocking, samples were incubated in the dark for 1 h at 4 °C with a saturating concentration of anti-human primary mouse monoclonal antibodies. After two washes in PBS 1X +1% BSA, cells were incubated with secondary antibodies: Alexa Fluor 488^® F(ab’)2 fragment of goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) and/or Alexa Fluor 594^® F(ab’)2 fragment of goat anti-mouse IgG (H + L) (Invitrogen). The samples were then washed with PBS 1X + 1% BSA and resuspended in 200 μL of the same buffer. The macrophages were electronically gated according to light scatter properties to exclude cell debris and contaminating lymphocytes. Fluorescence was measured using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OH, USA).

Primary antibodies: The following primary antibodies were used (antigen, dilution, vendor): Sel1L, 1:1000, Abcam ab78298; Actin, 1:4000, Sigma A5441; NPC1, 1:500 western, Abcam ab36983 (discontinued); NPC1, 1:200 imaging, Abcam ab134113; Flag, 1:1000, Sigma F1804; GP78, 1:1000, Cell Signaling 9590; Der-1, 1:1000, Cell Signaling 8897; HRD1, 1:1000, Cell Signaling. D302a; HA, 1:1000, Biolegend 901501; LC3, 1:1000 western, Novus NB600; LC3, 1:200 imaging, Gentex GTX17380; Beclin-1, 1:1000, Santa Cruz H-300; P97, 1:1000, Cell Signaling 2648; Ubiquitin, 1:500, Dako (discontinued); GAPDH, 1:5000, Novus NB600; Vinculin, 1:2000, Sigma V9131; FAM134B, 1:500, Abcam ab151755; FAM134B, 1:500, Proteintech 21537; p62, 1:1000, Sigma P0067; GBA, 1:1000, Sigma G4171; Calreticulin, 1:1000, Cell Signaling Technology 2891, GFP, 1:1000, Abcam ab13970. NPC1, 1:500, Abcam ab134113 was used for Figs. 3b and 5c, Supplementary Fig. S1b, c.
Secondary antibodies: The following secondary antibodies were used (antibody, dilution, vendor): Alexa Fluor 488 Goat anti-rabbit IgG (H+L), 1:500, Invitrogen A11008; Alexa Fluor 594 Fab’2 fragment of goat anti-mouse IgG (H+L), 1:500, Invitrogen A11020; Alexa Fluor 594 goat anti-chicken IgY (H+L), 1:1000, Invitrogen A11042; Goat anti-mouse IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6516; Goat anti-rabbit IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6515.