Rabbit anti gfp antibody

To investigate the interaction between LvMALT1 and LvTRAF6, HA-tagged LvMALT1 was co-transfected with LvTRAF6-GFP or GFP (as a control) into S2 cells. Additionally, LvTRAF6-GFP was co-transfected with three truncated forms of LvMALT1 (1–100 aa, 101–300 aa, 301–787 aa), each tagged with HA. After 48 h of plasmid transfection, cells were collected and lysed using IP Lysis Buffer (Pierce; cat. no. 87788) supplemented with a Halt Protease Inhibitor Cocktail (Thermo, Waltham, MA, USA, 87788). Approximately 90% of the cell lysate was incubated with agarose affinity gel containing anti-GFP (MBL International Corporation, Woburn, MA, USA, D153-8). The remaining 10% of the cell lysate was used as input for reference. All samples underwent SDS-PAGE assays. For Western blotting, we used rabbit anti-GFP antibody (Sigma-Aldrich, St.Louis, MO, USA, SAB4301138) as the primary antibody, and anti-rabbit IgG HRP-conjugate (Promega, Madison, WI, USA, W401B) as the secondary antibody. All antibodies were diluted in TBS.

To explore the potential interaction between LvFLp53, LvΔNp53 and LvDorsal, pAc5.1-LvFLp53-FLAG or pAc5.1-LvΔNp53-FLAG was transfected with pAc5.1-LvDorsal-GFP or pAc5.1-GFP (as a control) into S2 cells. After 48 h, cells were harvested and lysed in Pierce IP lysis buffer (Thermo, USA) with proteinase inhibitor cocktail (Sigma, USA). The co-immunoprecipitations were performed using anti-FLAG tag agarose conjugated gel (Abmart, China) and anti-GFP tag agarose affinity gel (MBL International Corporation, Japan), respectively. Western blotting was performed with rabbit anti-GFP antibody (Sigma, USA) and rabbit anti-FLAG antibody (Sigma, USA), and alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (Sigma, USA). A standardized aliquot (5%) of each total input cell lysates was also examined as control.

Co-immunoprecipitation (Co-IP) assays were performed as described in the previous study (36 (link)). To investigate the interaction of LvGSK3β with LvDorsal or LvCactus, pAc-LvGSK3β-GFP or pAc5.1-GFP (control) were co-transfected with individual plasmids along with pAc-LvDorsal-HA or pAc-LvCactus-HA into the S2 cells. Forty eight hours post transfection, cells were harvested and washed with ice-cold PBS three times, and then lysed in IP Lysis Buffer with a protease inhibitor cocktail (Sigma, USA). Both Co-IP and reciprocal Co-IP experiments were carried out using anti-HA affinity gel (Sigma, USA) and samples were subjected to SDS-PAGE assay. The precipitated protein was examined using western-blots, with rabbit anti-GFP antibody as the primary antibody, and alkaline phosphatase-conjugated goat anti-rabbit as the secondary antibody (Sigma, USA). A standardized aliquot (5%) of each total input cell lysates was also examined as control.

MUC1-∆CT-expressing U2OS cells were detached non-enzymatically with a cell dissociation buffer (Gibco, #13151014) at 37 °C for 15–20 min. After three gentle washes with an ice-cold FACS buffer (0.5% BSA in 1X PBS), cells were stained with rabbit anti-GFP antibody (Sigma-Aldrich, #PC408) at 1:500 dilution in the FACS buffer for 30 min on ice. After washing with the ice-cold FACS buffer, cells were then labeled with goat anti-rabbit IgG Alexa Fluor 647 (Invitrogen, #A-32733) at 1:1000 dilution for 30 min on ice in the dark. Free antibodies were then removed by an ice-cold 2 mM EDTA in the FACS buffer. Sytox Blue (Invitrogen, #S34857) was then added to cells to check cell viability. A MACSQuant flow cytometer (Miltenyi Biotec) was used for the analysis. The raw data were further processed using FCS Express™ 7 (De Novo Software).

WT (Lhx2f/f:TCA-GFP) and cKO (Lhx2f/f:NexCre:TCA-GFP) mice were transcardially perfused with 4% phosphate-buffered PFA at P7, and then postfixed in the same solution for 1 d. Brains were then cryoprotected with 30% sucrose in PBS, embedded in Tissue Tek OCT compound (Sakura Finetek), and cut in 20-25 µm sections on a cryostat (Leica). Coronal sections were immunostained with rabbit anti-GFP antibody (Millipore) and Alexa-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Fluorescent images were acquired using a LSM880 confocal microscope (Zeiss) with the scaling of 0.647 µm × 0.647 µm per pixel.
For VGlut2 immunohistochemistry, after the brain was perfused with PBS, fixed with 4% PFA and cryoprotected with 30% sucrose in PBS as described above, it was cut parallel to the surface at 300-μm-thick sections using a vibratome. Subsequently, rabbit anti-VGlut2 antibody (Synaptic Systems) (1:500) and Alexa-conjugated secondary antibodies (Thermo Fisher Scientific) (1:1000) were applied on free-floating sections. The slices were incubated for 2 d in the primary antibody at 4°C and after thorough washing with PBS, for 4 h in the secondary antibody at room temperature. They were then mounted on slides and covered with Fluoromount medium (Thermo Fisher Scientific) before coverslipped and imaged at a confocal microscope.

Mice were anesthetized with isoflurane (Baxter Healthcare Corporation) and perfused intracardially with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄/ 2H₂O, 1.4 mM KH₂PO₄; all from Sigma-Aldrich) and 4% paraformaldehyde (PFA, Merck) in PBS prior to decapitation. Brains were removed and after post-fixation in ice-cold 4% PFA for 2 h each brain was embedded in 2.5% agarose (Invitrogen)/PBS. agarose embedded brains were sliced (70 μm sections) on a vibratome (VT1000s, Leica). To increase the contrast of the Venus signal in the thick vibratome sections, the Venus fluorescence was visualized together with its immunoreactivity by using a rabbit anti-GFP antibody (1:5,000, Millipore) and FITC-coupled anti-rabbit secondary antibody (1:200, Jackson ImmunoResearch). For the NeuN staining, mouse anti-NeuN antibody were used (1:1,000, Merck Millipore) and followed with Cy3-coupled anti-mouse secondary antibody (1:200, Jackson ImmunoResearch). Immunstaining was performed as described (Krestel et al., 2001 (link)).