Donkey α rabbit cy3

Two series of the brain sections were separately analyzed for oxytocin and pSTAT3 expression using immunofluorescence protocol optimized for the visualization of pSTAT3 [see (Levin et al., 2004 (link)) for details]. In brief, slides were blocked in 4% NDS, 0.4% Triton and 1% BSA in KPBS for 20 min and then incubated in rabbit-α-pSTAT3 (1:500; 9145 from Cell Signaling Technology) or mouse-α-Oxytocin (1:1000; Cat # MAB5296; Lot # 3061232; Chemicon; Temecula, CA, United States) in 1% NDS, 0.4% Triton and 1% BSA in KPBS for two overnights at 4°C. After primary incubation slides were rinsed in KPBS, followed by a secondary incubation step in donkey-α-rabbit–Cy3 or donkey-α-mouse–Cy3 (both 1:100; Jackson ImmunoResearch, Laboratories) in 1% NDS and 0.3% Triton in KPBS for 2 h at room temperature protected from light. Slides were rinsed again in KPBS, counterstained in DAPI for 4 min and washed in KPBS. After 30 min air drying slides were cover-slipped with Vectashield^® (Vectashield^® Hardset™; Antifade Mounting Medium for Fluorescence; Ref # H-1400; Lot # ZH0114). Slides were stored at 4°C in the dark until further analysis.

Neurons were fixed in 4% paraformaldehyde for 10 min and washed four times with phosphate-buffered saline (PBS). Permeabilization of the membrane was performed by adding 0.1% Triton X-100 (Sigma-Aldrich, Darmstadt, Germany) in a blocking solution (PBS with 0.1% BSA and 5% goat serum) for 4 min. After washing once with PBS, a blocking solution was added to the coverslips for 15 min. Primary antibodies were added, and the neurons were incubated for one hour at room temperature. The coverslips were washed three times with PBS and incubated at room temperature for one hour with the secondary antibodies. After washing three times with PBS, the coverslips were mounted in Fluoromount (Sigma-Aldrich, Darmstadt, Germany). The primary antibodies used for immunostaining were: mouse α-FGF14 (1:500; clone N56/21, catalog #75 096, lot 413-8RR-61, NeuroMab, Davis, CA, USA); and rabbit α-MAP2 (1:1000; catalog #AB5622, lot 2795016, Millipore, Burlington, MA, USA). The secondary antibodies used were donkey α-mouse Alexa488 (1:1000, Jackson, Lansing, MI, USA, 715-545-150) and donkey α-rabbit Cy3 (1:1000, Jackson, Lansing, MI, USA, 711-165-152).

Retigabine (RTG) (10 μM) was added to hippocampal neurons for 24 h (for VGlut staining) or 48 h (for VGAT staining) prior to fixation, meaning that the cells (control and treated) were at the same age when fixed for immunostaining (13 DIV). Neurons were fixed in 4% paraformaldehyde for 10 min and washed four times with phosphate buffered saline (PBS). Permeabilization of the membrane was performed by adding 0.1% Triton X-100 (Sigma) in a blocking solution (PBS with 0.1% BSA and 5% goat serum) for 4 min. After washing once with PBS, blocking solution was added to the coverslips for 15 min. Primary antibodies were added, and the neurons were incubated for 1 h at room temperature. The coverslips were washed three times with PBS and incubated at room temperature for 1 h with the secondary antibodies. After washing three times with PBS, the coverslips were mounted in Fluoromount (Sigma). The primary antibodies used for immunostaining were: rabbit α-Kv7.3 (1:150, Alomone Labs), mouse α-AnkyrinG (1:500, Neuromab), mouse α-FGF14 (1:500, Neuromab, 75096), rabbit α-MAP2 (1:1000, Millipore), rabbit α-VGAT (1:500, Synaptic Systems, 131002), guinea pig α-VGlut (1:500, Millipore, AB5905). The secondary antibodies used were: donkey α-mouse Alexa488 (1:1000, Jackson, 715-545-150), donkey α-rabbit Cy3 (1:1000, Jackson, 711-165-152), and donkey α-guinea pig Cy3 (1:1000, Jackson, 706-165-148).

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS, Bodinco DV), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. BHK-21 cells were cultured in Glasgow’s minimal essential medium (GMEM, Gibco) supplemented with 8% (v/v) FCS, 10% (v/v) tryptose phosphate broth (TPB), 10 mM HEPES pH 7.4, 100 U/ml penicillin, and 100 mg/ml streptomycin. MARC-145 were cultured in DMEM supplemented with 8% (v/v) FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. DMEM and cell culture supplements were obtained from Lonza.
Primary antibodies that were used were mouse-α-β-actin (A5316, Sigma-Aldrich), rabbit-α-GFP [9 (link)], mouse-α-V5 (37–7500, Invitrogen), mouse-α-myc (9E10, Roche), mouse-α-HA (ab18181, Abcam), and mouse-α-FLAG (F1804, Sigma-Aldrich). As secondary antibodies, goat-α-mouse-HRP (P0447, Dako), swine-α-rabbit-HRP (P0217, Dako), goat-α-mouse-AlexaFluor488 (A11001, Life Technologies), and donkey-α-rabbit-Cy3 (711-165-152, Jackson). The antibody α-ORF7 (P3-05-A27, MSD Animal Health) was obtained using mouse antiserum. The antibody α-nsp2/3 LV was obtained using rabbit antiserum [13 (link)].

We assessed the effect of POGLUT1 activity toward the development of indirect flight muscles in Drosophila (Gildor et al, 2012). Rescue experiments were performed with fly transgenes expressing human POGLUT1^wt (wild type) and human POGLUT1^D233E. w¹¹¹⁸ (wt), rumi^79/79, Mef2‐GAL4/UASattB‐POGLUT1^WT‐FLAG; rumi^79/79, and Mef2‐GAL4/UASattB‐POGLUT^D233E‐FLAG; rumi^79/79 animals were raised at 18°C until puparium formation. Pupae 0–1 h after puparium formation (APF) were incubated at indicated temperatures until 25% pupal development. Pupal indirect flight muscles were dissected and stained using standard methods. Antibodies are rabbit α‐Twist 1:5,000 (Roth et al, 1989), mouse 22C10 1:50 (DSHB), mouse α‐FLAG M2 1:100 (Sigma), donkey‐α‐rabbit‐Cy3 1:500, donkey‐α‐mouse‐Cy5 1:500 (Jackson ImmunoResearch Laboratories). Confocal images were scanned using a Leica TCS‐SP5 microscope and processed with Amira5.2.2. Myotube lengths were measured using a two‐dimensional measurement tool in Amira5.2.2. Mean myotube lengths and standard deviations were calculated for rumi^79/79 pupae with no rescue and rumi^79/79 pupae overexpressing POGLUT1^WT (n = 22) or POGLUT1^D233E (n = 54). One‐way ANOVA with Tukey's multiple comparisons test was used to determine the P‐values. Images were processed with Adobe Photoshop CS5; figures were assembled in Adobe Illustrator CS5.