Alexa fluor secondary antibody

Dissected mouse intestinal tissues were fixed in 4% PFA, embedded in OCT, and snap frozen in liquid nitrogen. Washed slides were permeabilized and blocked simultaneously with 1% TritonX-100/PBS/1% BSA for 1 h at RT. Primary antibodies were applied for overnight staining at 4 °C in 1% BSA/PBS. Alexa Fluor secondary antibodies (Invitrogen) at a concentration of 1:500 were used to reveal staining. Slides were counterstained and mounted with Vectashield anti-fade DAPI-containing mounting medium (Vector Laboratories). Fluorescence images were acquired using an Eclipse TU2000-U microscope (Nikon). Organoid staining has been achieved following fixation in 4% PFA at RT, wash with PBS and subsequent simultaneous permeabilization, and blocking with 0.5% TritonX-100/PBS/5% BSA for 1 h at RT^{59 (link)}. Primary antibody staining was achieved overnight at 4 °C in 1% BSA/PBS. Alexa Fluor secondary antibodies (Invitrogen) at a concentration of 1:500 were used to reveal staining. Fluorescence images were acquired using a Spinning disk confocal microscope (Zeiss). Employed antibodies are listed in Supplementary Table 2.

For primary hippocampal neurons, cells were rinsed with PBS and fixed with 4% PFA for 10 min. Cells were permeabilized with PBST (PBS with 0.2% Triton X-100) for 10 min and blocked with 1% BSA in PBST for 30 min. Primary antibodies were incubated at 4 °C overnight. Antibody against Tau1 (Millipore, MAB3420, monoclonal) was used for immunostaining. The cells were washed three times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen). Then these cells were washed three times with PBS and coverslips were mounted on slide glasses. Images were taken by using LSM780 confocal microscopy. For Neuro2A, cells were washed with ice-cold PBS followed by fixation using 4% PFA/sucrose for 20 min. After 3 times washing with PBS, cells were permeabilized with PBST (PBS with 0.5% Triton X-100) for 15 min and blocked with 1% BSA in PBS for 1 h. Primary antibodies were incubated at 4 °C overnight. Cells were washed 3 times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen) for 1 h at room temperature. Cells were washed three times with PBS and coverslips were mounted on slide glasses. Antibodies against GFP (Abcam, ab6556, polyclonal), MT-CO1 (Abcam, ab14705, monoclonal), and Flag (Sigma, F1804, monoclonal) were used for immunostaining. Images were acquired by using 405, 458, 488, 561, 594, and 633 nm lasers.

Cortical and hippocampal cell cultures were fixed in 4% paraformaldehyde containing 4% sucrose for 1 h. Cultures were incubated with Hoechst Stain 33258 (3 μg/mL) (Invitrogen) diluted in phosphate buffered saline (PBS, pH 7.4) for 5 min then rinsed with PBS and permeabilized in 0.2% Triton X-100 in PBS for 5 min. Cultures were then blocked with 5% bovine serum albumin in PBS for 20 min and incubated with primary antibodies overnight at 4°C. Primary antibody solutions included guinea pig anti-MAP2 (1:800, Synaptic Systems, Göttingen, Germany), rabbit anti-vGLUT1 (1:1000, Synaptic Systems), mouse anti-vGAT (1:500, Synaptic Systems), rabbit anti-synapsin (1:500, Millipore, Billerica, MA) or mouse anti-synaptophysin (1:500, Dako, Carpinteria, CA). After extensive rinsing with PBS, cultures were incubated with Alexa-Fluor® secondary antibodies (1:500, Molecular Probe, Eugene, OR) for 2 h at room temperature. After washing with PBS to remove unbound secondary antibody, wells were filled with PBS at 4°C, sealed tightly with Parafilm® (Bemis, Neenah, WI) and shipped to the U.S.E.P.A. laboratories in Research Triangle Park, NC under refrigeration.

Cells were seeded the day before the staining. The next day, cells were rinsed three times with PBS then fixed with ice cold methanol 100% for 10 minutes at -20°C. After three washes with PBS, cells were blocked in Normal Goat Serum 5%, for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C, and after three washes with PBS, secondary antibodies were incubated 3h at room temperature in the dark. After three washes with PBS, cells were incubated with DAPI (Molecular Probes NucBlue Live ReadyProbes Reagent R37605) as manufacturer protocol, then washed and mounted on slides with Mowiol 20%. All the antibodies were diluted in blocking solution. The following primary antibodies were used: NPM [25 (link)], SBDS (Santa Cruz S15 SC49257 1:25), eIF6 [58 (link)] (1:100), Lamp1 (Santa Cruz sc-20011 1:100). The following secondary antibodies were used: donkey anti-goat, donkey anti-mouse, donkey anti-rabbit (Alexa Fluor secondary antibodies, Molecular Probes 1:500). The cells were examined by confocal microscopy (Leica SP5) and analyzed with Volocity 6.3 software (Perkin Elmer). Immunofluorescence experiments were performed at least three times, in triplicate.