Tcs sl

In the IHC experiments, samples of liver tumors (T) were fixed in 4% paraformaldehyde and embedded in paraffin. The sections of 3 µm were incubated with rabbit polyclonal anti-IRS-4 (Upstate Biotechnology, Lake Placid, NY, USA), as described previously [2 (link)]. Immunostaining of the PCNA, Ki-67, and pH3 antigens in the histological sections was performed using routine techniques performed in the Pathology Department of the Hospital Central de la Defensa Gómez Ulla using the following antibodies PC10, SP6, and PHH3, respectively.
In the ICC experiments, HepG2 cells were cultured on glass coverslips and for some experiments were transfected as described above. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Then, HepG2 cells were incubated with anti-IRS-4 (1/100), pERK1/2, and anti-integrin α2 (1/100) rabbit polyclonal antibodies, or antibody-free PBS as a nonspecific control. After incubation, cells were labeled using FITC-conjugated anti-rabbit Ig antibodies (1/1000 dilution) (Molecular Probes, Eugene, OR). Nuclei were stained by incubation with propidium iodide (Molecular Probes, Eugene, OR) and the HepG2 cells examined under a laser scanning confocal microscope (Leica TCS-SL). In another set of experiments, F-actin was labeled using Phalloidin-FITC.

To obtain retinal sections for immunofluorescence analysis mouse eyecups were fixed, infiltrated in sucrose or acrylamide, embedded in OCT and cryosectioned as described [34] (link). Sections were incubated with blocking solution (3% normal goat serum, 1% BSA, 0.3% Triton X100 in PBS pH 7.4, 1 h at room temperature); primary antibody (14 h at 4°C), secondary antibody (1 h at room temperature), and fixed for 15 min in 4% paraformaldehyde prior to being mounted with Mowiol [Calbiochem, Billerica, MA]. An antigen retrieval treatment of retinal sections [incubation in 0.05 mg/ml proteinase K in PBS pH 7.4 for 2 min at room temperature followed by a heat shock at 70°C for 10 sec] was needed for GCAP2 immunostaining. Antibodies used were: a polyclonal anti-GCAP2 (35), monoclonal anti-GCAP2 [mAb2235, Millipore, Billerica, MA], rabbit monoclonal anti-14-3-3ε [EPR3918, abcam, Cambridge, UK]. Secondary antibodies for immunofluorescence were Alexa 488 goat anti-rabbit IgG and Alexa 555 goat anti-mouse IgG [Molecular Probes, Eugene, Oregon]. Images were acquired at a laser scanning confocal microscope (Leica TCS-SL and TCS-SP2).