Alexa fluor 633 goat anti mouse igg

Anti-FMRP (F6072) was purchased from US Biological (Swampscott, United States); anti-FMRP (ab17722), anti-FMRP (phospho S499) (ab48127), anti-calreticulin (ab4), anti-Lamin B1 (ab16048), anti-RPLP0 (ab88872), anti-CYFIP2 (AB95969), anti-nucleolin (ab22758), anti-MTC02 (AB3298), anti-nucleophosmin (ab10530), anti-ACAT1 (ab71407) and anti-EEA1 (ab2900) were purchased from Abcam (Cambridge, United Kingdom); anti-eIF5 (SC-28309), anti-N-WASP (sc-100964), and anti-Gα_q/11 (sc-392) were from Santa Cruz biotechnology (Texas, USA); anti-nucleoporin p62 (610498) and anti-Rac1 (#610651) were from BD Transduction (New Jersey, USA); anti-Histone H3 (# 9715) and anti-GAPDH (# 2118) were from Cell Signaling (Boston, USA) and anti-Actin (# MAB1501) from Millipore (Temecula, U.S.A). Anti-Na⁺/K⁺ ATPase (A276), anti-FLAG (F3165) and DAPI were obtained from Sigma-Aldrich. Alexa fluor 546 phalloidin and the secondary antibodies Alexa fluor 488 goat anti-rabbit IgG and Alexa fluor 633 goat anti-mouse IgG were obtained from Molecular Probes (Oregon, USA).

Immunostaining was then performed as previously described [32 , 33 (link)]. Mice were transcardially perfused with 4% paraformaldehyde (PFA), postfixed at 4 °C overnight, cryoprotected in 30% sucrose, and embedded in optimum cutting temperature (OCT) compound. Sections (25 μm thick) were obtained by a Leica cryostat (CM 3050S). Immunostaining was performed as previously described. Rabbit anti-calretinin (Millipore, AB5054, 1:1000), mouse anti-RFP (Abcam, ab125244, 1:500) and mouse anti-vGlut2 (Synaptic System, 135311, 1:500) were used as primary antibodies, and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, A11008, 1:500) and Alexa Fluor 633 goat anti-mouse IgG (Molecular Probes, A21050, 1:500) were used as secondary antibodies. Before coverslips were applied, the slides were incubated with DAPI (Sigma, D9564, 1:1000) for 15 min. Pictures were captured by a confocal microscope (Olympus, FV1000).
Images of dendritic arbors were captured under a 40 × objective lens with a confocal microscope (Olympus, FV1000) in Z-stack mode (Additional file 1) [34 (link)]. Neuron morphology was traced manually using the NeuronJ plugin in ImageJ software. Standard morphometric analysis (Sholl analysis) was conducted as described earlier. Significance was determined by a two-way repeated-measures analysis of variance (RM 2-ANOVA; genotype and circle radius as factors).

Immunostaining was conducted on isolated islets, fixed in 4% paraformaldehyde before being permeabilised using 0.1% Triton X-100 (Sigma). After blocking with 5% goat serum, islets were incubated overnight (4°C) with primary antibodies before incubation with secondary antibodies. Fluorescent staining was visualised using a laser-scanning confocal microscopy (BioRad) controlled by LaserSharp2000 (BioRad).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).

Mouse anti-occludin (1:120), mouse anti-claudin-4 (1:150), rabbit anti-ZO-1 (1:100), alexa fluor 488 goat anti-rabbit IgG (1:400), alexa fluor 633 goat anti-mouse IgG (1:400) and SlowFade diamond anti-fade mountant were purchased from Thermo Fisher Scientific, Inc. Each antibody was used at the dilutions described above for immunofluorescence-microscopy imaging experiments.

HCC cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1 Triton X-100 in PBS for 20 min. To denature the DNA, the cells were treated with 4 M HCl for 15 min at room temperature and neutralized with 100 mM Tris-HCl (pH 8.5) for 10 min. After washing with PBS, the cells were blocked with 5% BSA for 1 h. Then, the cells were incubated with primary antibodies overnight at 4 °C. After washing with PBS, the cells were incubated with secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG Abcam; Alexa Fluor 633 goat anti-mouse IgG, Thermo Fisher) and then stained with DAPI. The plates were scanned, and images were obtained with an Olympus IX83 microscope.