Alexa fluor 647 conjugated secondary antibody

The primary antibodies that were used for IFA staining are: Mouse anti-dsRNA (English & Scientific Consulting, Szirák, Hungary), Rabbit anti-dsRNA (Absolute antibody, Oxford, UK); Mouse anti-hnRNP K (Santa Cruz, California, USA); Goat anti-eIF3 (Santa Cruz, California, USA); Rabbit anti-G3BP2 (Bethyl Laboratories, Texas, USA). The corresponding secondary antibodies that were used for IFA staining are: Alexa Fluor 488-, Alexa Fluor 594-, and Alexa Fluor 647-conjugated secondary antibodies (Invitrogen, California, USA). The primary antibodies that were used for WB are: Rabbit anti-eIF4G (Bethyl Laboratories, Texas, USA); Mouse anti-tubulin (Sigma, Andalucia, Spain). The monoclonal rat anti-2A antibody was a kind gift from Malin Flodström-Tullberg. Respective IRdye680- or IRdye800-conjugated secondary antibodies (LiCOR, Lincoln, NE, USA) were used for detection.

Cultures were fixed with 4% paraformaldehyde (PFA; in PBS, pH 7.4) at 10 DIV, permeabilized with 0.1% Triton X-100 during 20 min, and nonspecific binding sites were blocked with 10% bovine serum albumin for 10 min. Primary antibodies were incubated overnight at room temperature: anti-TH (1:2000, AB152, Cedarlane), anti-MAP2 (1:2000, MAB3418, Millipore Sigma), anti-RFP (1:1000, 600-401-379, Cedarlane), anti-Syt-1 (1:200, 105102, Synaptic Systems). These were subsequently detected using Alexa Fluor-488-conjugated, Alexa Fluor-546-conjugated, Alexa Fluor-568-conjugated, or Alexa Fluor-647-conjugated secondary antibodies (incubated at room temperature at 1:1000 for 1 h, Invitrogen).

For Native-PAGE western blots, proteins were transferred to nitrocellulose membrane (GE) using Tris-glycine transfer buffer (Novex). Primary antibodies were purchased from Invitrogen (anti-oligomer A11, and anti-Aβ N-terminus clone NAB228) and diluted 1:1000 in TBST +5% nonfat milk prior to use. AlexaFluor-647 conjugated secondary antibodies (Invitrogen) were diluted 1:3500 in TBST prior to use. Membranes were blocked for 1 h at room temperature in TBST +10% nonfat milk, briefly washed with TBST, incubated with primary antibody for 1 h at room temperature, washed with TBST (3 × 5 min), incubated with secondary antibody for 1 h at room temperature, washed (3 × 5 min), and imaged on a Molecular Dynamics Typhoon 9410 Variable Mode Imager. Dot blots were performed by spotting protein on 0.1 μM nitrocellulose membranes and processed the same as Western blots.

For immunofluorescence confocal microscopy, three-dimensional structured illumination microscopy (3D-SIM), and electron microscopy, cells were processed as previously described^{32 (link)}. Briefly, cells on coverslips were treated with aphidicolin for synchronization at the G1/S phase transition and released with fresh medium at the indicated times. The cells were fixed in methanol and incubated with the indicated primary antibodies. After wash, the cells were incubated with Alexa Fluor 488-, Alexa Fluor 568-, or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen). In some experiments, cells were immunostained with the Alexa Fluor 568-conjugated CEP120 antibody. The samples were viewed on a confocal system (LSM 700 or LSM880 Airyscan systems; Carl Zeiss). The 3D-SIM super-resolution images were acquired using a Zeiss ELYRA system equipped with a Plan Apochromat 63 × /1.4NA oil-immersion objective and the ZEN software (Carl Zeiss). For electron microscopy (EM), cells were grown on Aclar film (Electron Microscopy Sciences), fixed, and embedded in Spurr’s resin as previously described^{32 (link)}. Thin sections (100 nm) were stained with 4% uranyl acetate and Reynold’s lead citrate and the samples were examined with an electron microscope (T FEG-TEM; FEI Tecnai G2 TF20 Super TWIN)^{32 (link)}.

Cells grown on coverslips were fixed in methanol at −20 °C for 5 min. To examine the astral microtubules, cells were pre-extracted with 0.5% Triton X-100 in PHEM buffer (120 mM PIPES, 50 mM HEPES, 20 mM EGTA, 8 mM MgSO₄) for 1 min at 37 °C then fixed in methanol at −20 °C for 5 min. The fixed cells were then blocked with 10% normal goat serum in PBS and incubated with the indicated primary antibodies. After being washed with PBST (PBS containing 0.1% Tween-20), the cells were incubated with Alexa Fluor 488-, Alexa Fluor 568-, or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen). DNA was counterstained with DAPI (4,6-diamidino-2-phenylindole). The samples were mounted in Vectashield mounting media (Vector Laboratories) and visualized on a confocal microscope (LSM 880; Carl Zeiss, Jena, Germany) with a Plan Apochromat 63x/1.4 NA oil-immersion objective. Images were acquired with the ZEN software (Carl Zeiss).

The HepG2.2.15 cells were grown in six well plates with cover slips. After 48 hours, cells were fixed with 4% paraformaldehyde for 15 min followed by incubation PBST with 0.5% Triton X-100 for 20 min. Cells were blocked by goat serum and then incubated with Id1 and E2F4 antibodies at room temperature for 1 h. Mouse anti-Id1 monoclonal antibody (sc-133104) and mouse anti-E2F4 monoclonal antibody (sc-511) was obtained from Santa Cruz Biotechnology (USA). Bound primary antibody was visualized by Alexa Fluor 488-conjugated secondary antibodies and Alexa Fluor 647-conjugated secondary antibodies (Invitrogen, USA). Besides, Id1 and E2F4 nuclear localization were detected by immunofluorescent assay in HepG2.2.15 cells. Cell nucleuses were stained with DAPI. Cells were observed with a FluoView FV1000 laser scanning confocal microscope (Leica, Germany).

Frozen sections were fixed with 4% paraformaldehyde for 15 min and then blocked with 2% bovine serum albumin for 1 h at room temperature. After blocking, sections were incubated with EIF3H (Cell Signaling, 3413, 1:2000) and HAX1 (Proteintech, 11266-1-AP, 1:500) antibodies at 4 °C overnight. The samples were subsequently incubated with Alexa Fluor 488-, Alexa Fluor 594- or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen, 1:800). DAPI was then used for counterstaining the nuclei. Fluorescence signals were imaged on the Zeiss LSM 710 Multiphoton Laser Scanning Confocal.