Lsm780 confocal laser scanning microscope system

To obtain frozen brain sections for immunofluorescent assay, rats with correct AVV infusion sites were injected intraperitoneally with chloral hydrate (4 mL/kg) at 24 hours after completion of behavioural tests. The heart was initially perfused with normal saline followed by a slow perfusion with 4% paraformaldehyde (PFA). The brain was removed and placed in 4% PFA at 4℃ overnight. The brains were then subjected to sucrose gradient sedimentation, embedding and sectioning on a frozen microtome to obtain brain slices for immunofluorescence assay (N = 6/group). After brain sections (30 μm) were equilibrated to room temperature for ten minutes, the slices were placed in an immunofluorescent blocking solution and incubated for 60 minutes at room temperature. The primary antibody (1:100) was added to the brain slices and incubated at 4℃ overnight. After washing 3 times with PBS buffer, the fluorescent secondary antibody (1:200) was added and incubated for 60 minutes at 37℃ in a constant temperature shaker (avoiding light). The slices were then washed 3 times with PBS buffer, 75% glycerine sealed and then viewed as soon as possible. Immunofluorescent stained images were acquired with use of a LSM780 laser scanning confocal microscope system (ZEISS). At least six representative images were obtained from each rat for analysis by Image‐Pro plus 6.0 software.

V. carteri transformants expressing YFP-tagged Vc2c-Cyclop1 were examined using an inverted LSM780 confocal laser scanning microscope system (Carl Zeiss GmbH, Germany) and the ZEN digital imaging software (ZEN 2011, Carl Zeiss GmbH, Germany). Excitation was performed using the 514-nm emission line of an argon ion (Ar⁺) laser. YFP was detected between 520 and 550 nm [60 (link)], and chlorophyll was detected between 650 and 700 nm. Transmission images were obtained by using a transmission-photomultiplier tube (PMT) detector.