Mouse anti rab5

Mouse ES cells were provided by the Crick Institute Biological Resource Unit and were derived from hybrid blastocysts generated by the mating of C57BL/6J and 129 (S6)SvEv mice as previously described (29 (link)).
Unlabeled l-arginine and l-lysine, (R0K0, Sigma-Aldrich, Gillingham, United Kingdom), medium l-arginine [¹³C₆] and l-lysine [D₄] R6K4) and heavy l-arginine [¹³C_6,¹⁵N₄] and l-lysine [¹³C_6,¹⁵N₂] (R10K8) in their hydrochloride forms were obtained from CK Isotopes (Ibstock, United Kingdom).
Rat antihemagglutinin (HA; Roche, Burgess Hill, West Sussex, United Kingdom; 1:1000), rabbit anti-Arl8b (Biorbyt, Cambridge, United Kingdom; 1:200), goat anti-LIMP2 and anti-CAR (R&D Systems, Abington, United Kingdom; both 1:100) primary antibodies were used for immunofluorescence. For western blots, we used the following: mouse anti-Rab5 (Synaptic Systems, Göttingen, Germany; 1:500), rabbit anti-Rab7 (Cell Signaling Technology, Leiden, The Netherlands; 1:1000), rabbit anti-Arl8b (1:500) (30 (link)), goat anti-CAR (R&D Systems, 1:500) and mouse anti-βIII-tubulin (Covance, London, United Kingdom; 1:1000).

At DIV 7, cultured primary neurons in 24 wells were washed once with 1X PBS, then fixed in 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 10 min. After three times washing with 1X PBS, cells were blocked with 10% normal donkey or goat serum in 1 X PBS for 30 min at RT followed by three times washing in 1 X PBS. Thereafter, cells were incubated with primary antibodies diluted in 1 X PBS containing 1% normal donkey or goat serum for 2–3 hr at RT. three times washing with 1 X PBS, incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (1:500, Invitrogen) in 1 X PBS containing 1% normal donkey or goat serum for 1 hr at RT. Washed with 1 X PBS for three times, then counterstained the slides with DAPI (1:2000, Sigma) and mounted by using Vectashield (Vector) after rinsing. Primary antibodies used in this study were rabbit anti-APP (1:100, Synaptic Systems, 127 003), mouse anti-rab5 (1:100, Synaptic Systems, 108011), mouse anti-rab11a (1:20, Santa Cruz, sc-166523), mouse anti- Golgin-97 (1:100, Invitrogen, A-21270), rat anti-Lamp1 (1:20, Santa Cruz, sc-19992). After staining, images were obtained by using confocal microscope (Olympus FV-1200 or Leica SP8). The percentage of APP or APP-ΔCRD co-localizing with rab5, rab11, Golgin-97 and Lamp1 was calculated using JACOP (Bolte and Cordelières, 2006 (link)).