Alexa fluor 568 conjugated anti mouse igg antibody

HepG2 cells were seeded at 50 000 cells/well and A549 cells at 30 000 cells/well in 24-well plates 24 h before incubation with HFCP, pectin or etoposide for 24 h. After incubation, the cells were fixed and permeabilized for 10 min with a cold solution of 80% methanol and 20% acetone, rinsed three times with PBS-2% BSA and incubated for 2 h with primary antibodies. The primary antibody for LC3 staining was rabbit anti-LC3 (L7543 Sigma) (1/250 dilution), and the primary antibody for LAMP1 staining was mouse anti-LAMP1 (H4A3 received from August & Hildreth, Baltimore [13 (link)]). The cells were washed three times with PBS-2% BSA and then incubated for 1 h with the secondary antibodies. Alexa Fluor-488-conjugated anti-rabbit IgG antibody and Alexa Fluor-568-conjugated anti-mouse IgG antibody (Molecular Probes) were used at 1/1000 dilution. After 1 h of incubation, the cells were rinsed three times with PBS. For nuclear labeling, the cells were incubated for 30 min with TOPRO–3 (Molecular Probes, dil. 1/80) in the presence of 2 mg/ml RNAse and then rinsed three times with PBS. Finally, coverslips were mounted using Mowiol (Sigma, St Louis, USA), and the cells were observed with a fluorescent confocal microscope (Leica SP5).

We prepared the hippocampal slices (350 μm thickness) from postnatal day 7 Sprague Dawley rats and kept them in culture for 7–13 days in a CO₂ incubator at 34°C, as described previously in other studies [14 (link),32 (link)]. Concentrated Lv-Kir2.1 solution was injected into the extracellular space of the pyramidal cell layer of the slice culture with Femtojet® and Femtotips II ® (Eppendorf, Hamburg, Germany). The slices were incubated for 7 days until fixation. The fixed slices were counterstained with anti-NeuN antibody (MAB377; Millipore, Billerica, MA, USA) and a secondary antibody (Alexa-Fluor-568-conjugated anti-mouse IgG antibody; Molecular Probes, Eugene, OR, USA) to visualize the neurons.