63 oil immersion objective lens

Hippocampal neurons cultured for 3 days were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.4% Triton X-100 in PBS. The samples were incubated with PBS containing 10% donkey serum for 1 h at room temperature and then were incubated with the primary antibodies: rabbit anti-GFP (1:500, Invitrogen), goat anti-TrkB (1:500, R&D, Minneapolis, MN, USA, AF1494), mouse anti-GFP (1:200, Millipore, Temecula, CA, USA, MAB3580), or rabbit anti-JNK3 (1:500, Millipore, 04-893) at 4 °C overnight. Samples were washed three times with PBS and incubated with fluorescent secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) for 1 h at room temperature. All of the images were captured with a Zeiss LSM780 confocal microscope fitted with a × 63 oil-immersion objective lens (Microstructural Platform of Shandong University).

Localization of endogenous transgelin in RKO, SW480, HCT116 and LOVO cell lines and the expression of PARP1 in RKO cells were determined by immunofluorescence. The primary antibody (anti-transgelin, 1:500, Abcam, USA; anti-PARP1, 1:500, Cell signaling technology, USA), secondary antibody (Alexa Flour 594 goat anti-rabbit IgG, Alexa Flour 488 goat anti-rabbit IgG, 1:500, Invitrogen, USA), and the VECTASHIELD mounting medium (Vector Laboratories, USA)) with 4′, 6-diamidino-2-phenylindole (DAPI) were used. The immunofluorescence images were taken and preserved under the laser scanning confocal microscope using a 63 × oil-immersion objective lens (Carl Zeiss, USA).

The slides were analysed using a Zeiss 510 confocal microscope using the 63× oil-immersion objective lens (Zeiss, Oberkochen, Germany). Fluorescence was excited at 405, 488, and 561 nm to image DAPI, AlexaFluor488, and Cy3, respectively. Fluorescent emissions from fluorophore-conjugated secondary antibodies were acquired by sequential scanning using the Zen 2013 software.