Alexa fluor 647 goat anti rabbit igg

To demonstrate whether the temperature rise during PACT clearing influences the binding of antibody and antigen, the immunostaining was performed on 1-mm-thick Thy1-GFP-M brain section referring to the original PACT method31 (link). Before staining, the 1-mm-thick slices were cleared with 8% SDS at 37 °C, 42 °C, and 47 °C, respectively. Then the previously cleared samples were immunostained for parvalbumin and nuclei stained with DAPI. After washed in PBST for 1 day and blocked in PBS/0.1% Triton X-100/6% goat serum for 1 day, the samples were transferred to primary antibody dilutions (anti-parvalbumin antibody, Abcam, ab11427, 1:400) for 1 day followed by washing with PBST for several times, then to secondary antibody dilutions (Alexa Fluor 647 goat anti-rabbit IgG, Jackson Immunoresearch, 111-607-003, 1:400) for 1 day at 37 °C. Then the samples were nuclei stained with DAPI at room temperature for 12 hours. The samples were finally washed in PBST for several times before further incubating in 70% (wt/vol) sorbitol solution for 1 or 5 hours. For GFP immunostaining, the cleared brain slice was incubated in primary antibody dilutions (anti-GFP antibody, Millipore, AB3080, 1:200) for 2 days and secondary antibody dilutions (Alexa Fluor 633 goat anti-rabbit IgG, Invitrogen, A-21070, 1:200) for 2 days.

Cells were fixed in 70% ethanol and stained with rabbit anti-Histone H3 (phospho S10) antibody (ab5176, Abcam) for 1 h, followed by 30 min of incubation with Alexa Fluor 647 goat anti-rabbit IgG (Jackson). DNA was stained with Hoechst 33242 (1.5 µg ml⁻¹). Flow cytometry analysis was performed on LSRII flow cytometer (BD Biosciences) using FACS Diva (BD Biosciences) software and data have been analyzed using FlowJo software. For the percentage of multinucleated cells within the population, cells containing >4N DNA content have been gated.