Alexa 555 donkey anti rabbit

Fluorochrome-conjugated antibodies used: anti-CD11c-APC, anti-IFNγ-APC, anti-TNFα-PE-cy7, anti-IL-4-PE, anti-IL17-PE, anti-Foxp3-PE (e-Bioscience, Vienna, Austria), and anti-LAMP-1-v450 (BD-Pharmingen). Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le^X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova). Secondary antibodies used: peroxidase-labeled F(ab’)₂ fragment goat anti-human IgG, F(ab’)₂ fragment goat anti-mouse IgG, (Jackson), peroxidase-labeled goat anti-mouse IgM (Nordic Immunology), goat anti-rat Alexa 448, goat anti-rat Alexa 647, donkey anti-goat Alexa 488, donkey anti-goat Alexa 647, donkey anti-rabbit Alexa 555 and donkey anti-rabbit Alexa 488 (Molecular Probes). MGL-1-Fc was generated as described earlier (Singh et al., 2009b (link)). MR-Fc was kindly provided by L. Martinez-Pomares (University of Nottingham, Nottingham, UK).

COS7 cells were seeded on to glass coverslips and after 24 h were co-transfected with pcDNA3.1 PPCA and pIRES NEU1-hrGPF-1a (wild-type or V217A, D234N mutants) as indicated above. After 24 h transfection the medium was changed and after further 24 h cells were washed three times with PBS containing 1 mM MgCl₂ and 1 mM CaCl₂ (PBS⁺⁺), fixed and permeabilized with cold methanol for 10 min and acetone for 1 min. After three washes and saturation with 1% BSA in PBS⁺⁺ (PBS⁺⁺/BSA), glass coverslips were incubated with the following primary antibody: rabbit anti-NEU1, Rockland 1∶500, mouse anti-LAMP1, BD Pharmigen 1∶200, mouse anti-PDI, STRESSGEN 1∶200. Subsequently, cells were washed and incubated with the following secondary antibody: Donkey anti-rabbit Alexa-555 and goat anti-mouse Alexa-405 1∶300 (Molecular Probes, Invitrogen) diluted in PBS⁺⁺/BSA. Finally, specimens were mounted using Dako Cytomation Fluorescent Mounting Medium and analyzed using the confocal system LSM-510 META (Carl Zeiss). Images were processed with LSM Image Browser (Carl Zeiss) and Adobe Photoshop software.

Muscles were frizzed in OCT and kept at -80C. Immunocytochemistry was performed on slides of 4µm using Polyclonal Rabbit Anti-Laminin-1 (Dako) (1/1000) as primary antibody and Donkey anti Rabbit Alexa 555 (Invitrogen) as secondary antibody (1/1000). Images were acquired with IX83 Olympus microscope and ORCA/4 Hamamatsu camera at objective 10 with Mcherry filter. Muscle sizes were obtained after binary transformation with ImageJ 1.53c software (30 (link)).

Cells were carefully washed once with PBS. Fixation was carried out with 3.7% formaldehyde (Carl Roth) in PBS for 10 min at RT. Cells were then permeabilized with 3 mM sodium citrate tribasic dehydrate (Merck), 0.1% v/v Triton X-100. Permeabilized cells were washed twice with PBS and twice in washing solution [PBS, 0.1% v/v Tween 20, 0.2% w/v BSA] for 5 min. Cells were blocked with blocking solution [PBS, 0.1 % v/v Tween 20, 2.5% w/v BSA] for 30 min and incubated overnight at 4 °C with primary antibodies in blocking solution. The following antibodies were used: GFP (Aves, GFP1020, 1:1000), IAP-GAG (Cullen lab, 1:1000). Cells were washed three times in washing solution for 10 min before incubation with secondary antibodies Donkey anti Chicken IgY Alexa488 (Jackson Immuno Research, 703-545-155, 1:800) and Donkey anti rabbit Alexa 555 (Invitrogen, A-31572, 1:1000) in blocking solution containing 10% normal goat serum (Dianova-Jackson Immuno Research) at RT for 1 h. After washing three times in PBS, 0.1% Tween 20 for 10 min, cells were embedded with Vectashield/DAPI (Vector Laboratories) on standard microscope slides (Carl Roth). The immunofluorescence staining was examined with Axiovert 200 M inverted microscope for transmitted light and epifluorescence (Carl Zeiss Microscopy) with the help of the AxioVision Special Edition Software (Carl Zeiss Microscopy).