Alexa fluor 488 labeled goat anti rabbit antibody

Primary hippocampal neurons were fixed with 4% paraformaldehyde/4% sucrose for 15 min at room temperature, permeabilized with 0.2% Triton X-100 for 10 min, then blocked for 1 hr in 5% goat serum, followed by primary antibody incubation overnight at 4°C and secondary antibody incubation for 1 hr at room temperature. The following primary antibodies were used: Fyn (Cell Signalling Technologies #4023; rabbit polyclonal, 1:500), PSD-95 (Millipore, monoclonal, 1:500), MAP2 (Millipore, chicken polyclonal, 1:500), and phalloidin-Alexa Fluor 647 (NEB, 1:100), which was used to detect actin. As secondary antibodies, we used Alexa-Fluor-488-labeled goat-anti-rabbit antibody, Alexa-Fluor-555-labeled goat anti-mouse antibody and Alexa-Fluor-647-labeled goat anti-chicken antibody (all from LifeTechnologies, Thermo Fisher, 1:500). Fluorescence images were captured with a 20X or a 60X objective on a Zeiss LSM710 confocal microscope and analyzed with Fiji-ImageJ software (Schindelin et al., 2012 (link)).

Frozen specimens were sliced into 7 μm-thick sections, mounted on glass slides, and fixed in acetone for 10 min at room temperature (20–25° C). To block nonspecific binding sites, the slides were incubated with 2% goat serum in PBS for 30 min at room temperature (20–25° C). Slides were then incubated overnight at 4° C with the following primary antibodies: p16 (ab108349, Abcam, 1:200) and Ki67 (ab16667, Abcam, 1:200). After washing three times with PBS, slides were incubated at room temperature (20–25° C) with Alexa Fluor 488-labeled goat anti-rabbit antibody and Alexa Fluor 555-conjugated goat anti-mouse antibody (both from Thermo Fisher Scientific), diluted 1:1000 in PBS for 1 h. After washing three times with PBS, the samples were counterstained for nuclear visualization using ProLong Gold Anti-fade Mountant (Thermo Fisher Scientific) containing DAPI.

All chemicals used were purchased from Sigma Aldrich (Switzerland) unless otherwise indicated. All the primers (S1 Table) for real time PCR were purchased from Life Technology (Switzerland). The polyclonal antibodies against ATP8A1 was obtained from Proteintech (cat# 21565-1-AP, Rosemont, IL, USA). The polyclonal antibody against ATP11C (cat# ab175055) and the monoclonal antibody against RAB7 (cat# ab126712) were ordered from Abcam (Cambridge, MA, USA). The polyclonal antibody against ATP11A (cat# HPA035583) and the monoclonal antibody against ATP9A (cat# WH0010079M2) were obtained from Sigma (St. Louis, MO, USA). The monoclonal antibody against EEA1 is produced by BD Biosciences (cat# 610457, Franklin Lakes, NJ, USA). The HRP-conjugated goat-anti-rabbit antibody was purchased from GE Healthcare (cat# RPN4301, Little Chalfont, UK) and the HRP-conjugated goat anti mouse antibody from Pierce (cat# NA931V, Waltham, MA, USA). The Alexa fluor488 labeled goat-anti-rabbit antibody was from ThermoFisher Scientific (cat# A11008, Waltham, MA, USA) and the Cy3-conjugated affiniPure goat-anti-mouse antibody from Jackson ImmunoResearch (cat# 115-165-146, West Grove, PA, USA).