Lsm 800 laser

HeLa cells with fewer than 20 passages were maintained in Dulbecco’s Modified Eagles’ medium (Gibco) supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin–streptomycin (Gibco) at 37 °C under 5% CO₂ in humidified atmosphere. All constructs for cell studies were cloned into the pcDNA3.1(+) vector (Invitrogen). mCh-PRM-SH3-6His or mCh-PRM-SH3-transfected cells were treated with ZnCl₂ (1 μM) and incubated for 0, 5, 30, or 60 min at 37 °C. Cells were washed three times with DPBS and then fixed with 4% paraformaldehyde for confocal imaging. Fluorescence images were obtained with a LSM 800 laser scanning confocal microscope (Carl Zeiss, LSM 800) using 100× oil objective lens. Cell viability was examined by a tetrazolium-based calorimetric assay (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay). HeLa cells with fewer than 20 passages were seeded onto 96-well plates at a density of 1 × 10⁴ cells per well and incubated for 18 h. Transfected cells were treated with various concentration of ZnCl₂ and incubated in the medium for 6 h before a MTT analysis.

Larvae were fixed in 4% para-formaldehyde/PBS overnight at 4 °C. They were then rinsed in PBS. The brains were dissected out, and permeabilized using 1% BSA (fraction V; Sigma), 0.1% DMSO and 0.1% Triton X-100. The anti-GAD65/67 (Abcam ab11070, RRID:AB_297722; 1:500) has been previously used in zebrafish [70 (link)]. The brains were incubated in the primary antibody overnight, rinsed several times in PBS, then incubated in secondary antibody (Alexa 488 goat anti-rabbit; 1:1000). After washing, these were mounted in 1.2% agarose/PBS. Imaging was carried out using a Zeiss LSM 800 laser scanning confocal microscope, with a 40× water immersion objective.

HL7702, HepG2 and Hep3B cells were fixed with 4% paraformaldehyde. After the samples were blocked with 1% BSA, an anti-NEK2 mAb (1:500) was added for incubation. After incubation with FITC-labeled goat anti-mouse IgG (1:1000) as the secondary antibody, NEK2 subcellular localization was observed by a Zeiss LSM 800 laser confocal scanning microscope.
After HL7702, HepG2 and Hep3B cells were fixed with 4% polyformaldehyde, immunocytochemical staining was performed using a SP kit. Anti-NEK2 mAb (1:1000) as the primary antibody was added. In addition, EVOS software was used for observation. The expression of NEK2 mRNA in liver cancer cells was detected by real-time PCR, and total RNA of HL7702, HepG2 and Hep3B cells was extracted by TRIzol. Real-time PCR was performed according to the instructions of the reverse transcription kit. The results were analyzed by the 2^−△△CT method [9 (link)]. Finally, immunocytochemical staining results were compared with real-time PCR results. Moreover, we analyzed the expression levels of NEK2 between normal liver tissue and primary liver tumor tissue by the UALCAN database. UALCAN (http://ualcan.path.uab.edu/index.html) is a web-based tool that allows researchers to perform analyses of gene expression levels from The Cancer Genome Atlas (TCGA).