Alexa 568 conjugated anti mouse igg

Samples were harvested and resuspended in 4% paraformaldehyde (PFA) (Novagen, UK) and allowed to settle on poly-L-lysine- (Sigma, UK) coated slides overnight at 4°C. The slides were washed once with Tris-buffered saline (TBS) for 5 min and then permeabilized with 0.2% Triton X-100 (Sigma, UK) in PBS. The slides were then washed three times in TBS and incubated in blocking solution (10% goat serum (Sigma, UK) and 3% BSA (Sigma, UK) in TBS) for 45 min and subsequently probed overnight at 4°C with primary antibody in 1% BSA (Sigma, UK) in TBS. Primary antibodies were rabbit anti-myc polyclonal antibody (Cell Signaling, USA) and mouse anti-alpha tubulin (Sigma, UK). The slides were washed three times in TBS and incubated for 1 hr with secondary antibody in 1% BSA (Sigma, UK) in TBS. The secondary antibodies were Alexa-488 conjugated anti-rabbit IgG and Alexa568 conjugated anti-mouse IgG (Molecular probes, UK). The slides were washed three times in TBS and mounted in Vectashield with DAPI (Vector Labs). Preparations were labelled with secondary antibodies alone to verify the absence of non-specific labelling. Parasites were visualized on a Leica SP5 confocal microscope and acquired and analysed with the LAS AF Lite software (Leica, UK).

Immunocytochemical analysis was essentially performed as described previously [24 (link)]. Primary neurons cultured on coverslips were fixed with 4 % paraformaldehyde in PBS. Fixed cells were permeabilized and blocked with 0.3 % Triton X-100 and 1 % horse serum in PBS, and incubated with primary antibody for BACE1 (D10E5) or APP (22C11) for 1 h, followed by Alexa 488-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) for 1 h. For double immunofluorescence staining, cells were subsequently stained with anti-MAP2 antibody and Alexa568-conjugated anti-mouse IgG (Molecular Probes). Specimens were examined under an LSM 780 laser scanning confocal microscope (Carl Zeiss, Jena, Germany). The fluorescence intensities of neurites and soma were quantified according to previously documented methods [56 (link)]. Briefly, to quantify fluorescence intensity, 1 pixel-wide line segments were traced along 50 μm of neurites with soma as the starting point. The mean fluorescence intensity in soma was quantified by drawing a region around the soma. For each condition, ~20 cells from two different cultures were analyzed. To distinguish axons and dendrites, cells doubly immunostained with anti-BACE1 and anti-MAP2 were analyzed as above; an example image is shown in Additional file 4: Figure S4.