Goat anti rat alexa fluor 647

For immunostaining and imaging, A549 cells were cultivated on coverslips. The cells were fixed with 4% paraformaldehyde (PFA) for 20 min, permeabilized by 0.02% PBST (PBS with Triton X-100) for 20 min, washed 3X with PBS and blocked with 3% BSA in PBS for 30 min at room temperature. Anti-δ-hENaC antibody (1:500) was applied for 1 h at RT. After washing, cells were incubated with secondary antibody (goat anti-rat Alexa Fluor 647, Thermo Fisher Scientific), Vienna, Austria, for 1 h at RT in dark. Confocal images were acquired with a Zeiss LSM-510 confocal laser scanning microscope. Quantification was performed by ImageJ U.S. National Institutes of Health, Bethesda, Maryland, USA.

In immunofluorescence experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam, Cambridge, UK, cat # ab211324) 1:100; anti-SQSTM1/p62 (Abcam, cat # ab56416) 1:50; anti-β-Tubulin III (Merck Millipore, Burlington, MA, USA cat # T2200) 1:500; anti-MAP2 (1:500, Merck Millipore, cat # M9942) 1:500; anti-GFAP (Thermo Fisher Scientific, Waltham, MA, USA, cat # 13-0300), 1:800, donkey anti-rabbit-IgG Alexa Fluor 488 (Thermo Fisher Scientific, cat # R37118) 1:1000; donkey anti-mouse-IgG Alexa Fluor 594 (Thermo Fisher Scientific, cat # A-21203, 1:1000); goat anti-mouse IgG1 CF 568 (Merck, cat # SAB4600313 1:1000); goat anti-rat Alexa Fluor 647 (Thermo Fisher Scientific, cat # A21247, 1:1000); and DAPI (4′,6-diamidino-2-phenylindole Merck Millipore, cat #D9542) 1:5000. In Western blotting experiments, the following antibodies and dilutions were used: anti-Phospho SQSTM1/p62 (S349) (Abcam cat # ab211324) 1:2000; anti-SQSTM1/p62 (Abcam cat # ab56416) 1:2000; Anti-GAPDH (Abcam cat # ab8245); 1:5000; anti-phospho-Akt (Ser473 D9E Cell Signaling, Danvers, MA, USA, cat #4060) 1:2000; anti-Akt (Cell Signaling, cat # 9272, 1:1000); goat anti-mouse IgG IRDye 800(Li-Cor, Lincoln, NE, USA, cat # 926-32210) 1:5000; and goat anti-rabbit-IgG Alexa 680 (Thermo Fisher Scientific cat # A21076) 1:5000.

Adherent neurons were fixed in 4% paraformaldehyde for 5 min, followed by treatment with 0.5% saponin in PBS for 20 min for permeabilization. To block the samples, we treated the plates with 10% normal goat serum with 0.01% tween-20 in PBS for 30 min. Primary antibodies were then left incubating with the samples at 4°C overnight with 1% normal goat serum and 0.01% tween-20, before washing with PBS for three times. Secondary antibodies were then applied to 1% normal goat serum and 0.01% tween-20 at room temperature for 1 h before washing for another four times. The primary antibodies we used were Guinea pig anti-Synapsin I/II (Synaptic Systems, 1:500), rabbit anti-HOMER1 (Synaptic Systems, 1:500), chicken anti-MAP2 (Abcam, 1:1000), mouse anti-human nuclear antigen (Abcam, 1:200), rabbit anti-CUX2 (Abcam, 1:200) and rat anti-CTIP2 (Abcam, 1:500). The secondary antibodies we used were Goat anti-guinea pig Dylight 488 (Abcam), goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555, goat anti-chicken Alexa Fluor 555, goat anti-rabbit Alexa Fluor 647 and goat anti-rat Alexa Fluor 647 (Thermo) at 1:1000 dilution.

For immunofluorescence staining, cells or tumor tissues from each treatment group were washed with ice-cold PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 min at room temperature, followed by permeabilization in 0.2% Triton X-100-PBS for 10 min. Samples were followed by blocking with PBS blocking buffer containing 2% normal goat serum, 2% BSA, and 0.2% gelatin for 1 h at room temperature. Then, the samples were incubated in primary antibodies at the appropriate concentration for 1 h at room temperature, washed with PBS and incubated in goat anti-rat-Alexa Fluor 647 (Molecular Probes) at 1:1000 dilution in blocking buffer for another 1 h at room temperature. Finally, stained cells were washed with PBS, counterstained with Hoechst 33342 (Molecular Probes-Invitrogen, H1399, 1:10000 dilution in PBS), and mounted on slides with Prolong Gold antifade mounting medium (Life Technologies). The slides were imaged under a confocal laser scanning microscope (Olympus, FV1100).

The ovary and embryo immunostaining protocols used was previously described50 (link)51 (link). The primary antibodies used were as follows: mouse anti-GFP antibody (1:100; Roche), rabbit anti-Osk antibody (1:100; a gift from Dr. Tze-Bin Chou), rabbit anti-Tudor antibody (1:100; a gift from Dr. Akira Nakamura), rabbit anti-Krimp antibody (1:500; a gift from Dr. Toshie Kai) and rat anti-Vas antibody (1:100; Developmental Studies Hybridoma Bank). The fluorescently labelled secondary antibodies used were as follows: goat anti-mouse Alexa Fluor 488 (1:100; Invitrogen), goat anti-rabbit Alexa Fluor 647 (1:100; Invitrogen) and goat anti-rat Alexa Fluor 647 (1:100; Invitrogen).