Alexa 488 conjugated goat anti mouse antibody

For immunohistochemical studies, rats anesthetized with chloral hydrate (30 mg/kg, intraperitoneally) were perfused through the aorta with 100 ml 0.9% NaCl followed by 100 ml phosphate buffer containing 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde overnight then equilibrated in a cryoprotectant solution of 30% sucrose/PBS and stored at 4°C.
Immunohistochemistry and immunofluorescence staining were carried out as described previously [22 (link)]. The antibodies employed in the present study are listed as follows:
The primary antibodies used were I₁^PP2A mAb (5G6, 8.4 μg/ml), pAb Aβ34–40 (1:500; Invitrogen), pAb Aβ36–42 (1:500; Invitrogen), mAb GFP (1:500; Rockland), Tau mAb M4 to phosphorylated Thr 231/Ser-235 (1:1000; [24 (link)]), Tau mAb 12E8 to phosphorylated Ser 262/356 (1:250), mAb SMI52 to MAP2a,b (1: 1000; Covance), mAb synaptophysin (1:200; Chemicon, Temecula, CA, USA), and pAb synapsin-1 (1:500; Stressgen, Ann Arbor, MI, USA). Secondary antibodies used were: Alexa 488-conjugated goat anti-mouse antibody (1:1000) and Cy3 goat anti-rabbit antibody (1:1,000) (Jackson Immunoresearch, West Grove, PA). Finally, the sections were washed in TBS and mounted on glass slides with ProLong Gold antifade reagent (Invitrogen). Immunohistochemical analysis employed 3 animals/group.

Differentiated cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, and then permeabilized by 0.1% triton X-100 (Sigma) for 10 min. After washing and blocking, cells were incubated in the primary antibodies of NKX2.5 (Santa cruz, sc-14033), TNNT2 (DSHB, CT3), α-Actinin (Abcam, ab68167) or β-Catenin (Santa cruz, sc-59737) at 1:100 dilution in 1% BSA at 4 °C overnight. Cells were washed with PBS for three times, and then incubated with alexa 488 conjugated goat anti-mouse antibody (Jackson, 115-545-071) and/or alexa 594 conjugated donkey anti-rabbit antibody (Jackson, 711-585-152) at 1:500 dilution for 1 h at room temperature. After removing the secondary antibodies, the cells were incubated with DAPI for 10 min at room temperature. Finally, the samples were washed three times and then mounted with vectashield (Vector Laboratories). Images were taken using Carl Zeiss Confocal LSM710 or Leica confocal SP8. Sarcomere length was analyzed using Leica Application Suite X (LAS X).

Western Blot analysis was performed by SDS-page on cell lysates from A549 (lung carcinoma) and U87 (glioblastoma) cell lines exhibiting high PGP9.5 expression, to verify the accuracy of the rabbit polyclonal anti-human PGP9.5 antibody (RA95101, UltraClone Ltd., UK). Subsequently, western transfer of proteins was performed onto a transfer membrane (Immobilon FL, Li-Cor, Germany). After incubation of the rabbit polyclonal anti-human PGP9.5 antibody (1/300), visualization was performed using IRDye^® 800CW Conjugated Goat (polyclonal) anti-rabbit IgG antibody on the Odyssey^® Infrared Imaging System (Li-Cor).
For double immunofluorescence histochemistry, 16 µm-thick sections were incubated overnight at room temperature with a mix of primary anti-PGP9.5 (1/2000) and anti-βIII-Tubulin (clone 5G8, Promega Corp., USA; 1/250) antibodies to be subsequently visualized by Cy3-conjugated goat anti-rabbit and Alexa-488-conjugated goat anti-mouse antibodies (Jackson Immunoresearch Laboratories, Inc., USA; 1/500 and 1/100 respectively). Counterstain was performed using Hoechst 33342 (Invitrogen Molecular Probes, USA; 1/2000).

For the immunofluorescence (IF) assay, dHL60 cells were planted into a 15 mm glass-bottom cell culture dish (Corning, Corning, NY, USA) and treated with 10 μg/mL BifA protein for 2 h. Next, the cells were fixed with 4% paraformaldehyde (PFA) for 30 min after being rinsed twice with PBS. Fixed cells were permeabilized using 0.1% Triton X-100 and rinsed twice with PBS. The coverslips were blocked with blocking buffer (5% BSA and 0.1% Tween 20 in PBS) and incubated with mouse anti-BifA polyclonal antibody or rabbit anti-p-ezrin (T567) monoclonal antibody (Abcam) in blocking buffer for 1 h. For secondary antibodies, Alexa 594-conjugated goat anti-rabbit and Alexa 488-conjugated goat anti-mouse antibodies (Jackson Immunoresearch, West Grove, PA, USA) were diluted in blocking buffer. Finally, DAPI was used to stain the cell nuclei. A confocal laser scanning microscopy (Zeiss, LSM710, Jena, Thuringia, Germany) was used to acquire pictures from prepared samples. For densitometric analysis, pixel intensity was quantified using ImageJ software.