Axiocam 506 color microscope camera

HaCaT cells at a density of 0.5 × 10⁶ cells/well in DMEM were seeded onto glass coverslips placed in 3-cm dishes and cultured for 24 h. The medium was then replaced by FBS-free DMEM, and cells were cultured for 24 h. Cells were pre-treated with OXY at 40 µM for 3 h before stimulating with TNF-α for 40 min (for pAKT staining) and 20 min (for p-GSK3-_β staining). For Ki-67 and MCL-1, the OXY-treated cells were stimulated with TNF-α for 24 h. Cells were washed once with 1× PBS and fixed with 4% paraformaldehyde for 15 min. Sample coverslips were washed 3 times with PBS (5 min each time) and permeabilized with 0.3% TritonX-100 in PBS for 5 min. After washing trice, sample coverslips were blocked with 1% BSA in PBS for 1 h at RT, and incubated with primary antibodies in a moist chamber at 4 °C overnight. Sample coverslips were washed 3 times with 1× PBS and incubated with a secondary antibody conjugated with Alexa488 for 2 h in a moist chamber at RT. Sample coverslips were washed 3 times with 1× PBS and 1 time with deionized water (5 min) before mounting with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Signals of the target proteins were visualized at 100× magnification by a fluorescent microscope, Axio Vert.A1 (Carl Zeiss, Jena, Germany). The Zen 2.6 (blue edition) software for the Zeiss Axiocam 506 color microscope camera was used to capture and analyze the images.

Six isolates were identified with the use of the descriptions provided by Raper (1 ) and molecular characteristics proposed by Sheikh et al. (5 (link)). First, the location of each early aggregating clone and sorocarp that developed in a plate was marked. The characteristic stages in the life cycle, including cell aggregation and the formation of pseudoplasmodia and sorocarps, were observed under a Zeiss dissecting microscope (Axio Zoom V16) with a 1.5× objective and a 10× ocular. Slides with sorocarps were prepared with water as the mounting medium. Features of spores, sorophores, and sorocarps were observed and measured on the slides by using a Zeiss light microscope (Axio Imager A2), with 10× ocular and 10, 40, and 100× (oil) objectives. Photographs were taken with a Zeiss AxioCam 506 color microscope camera.

A total of 0.5 × 10⁶ cells/well of SKOV-3 were seeded onto glass coverslips placed in 3 cm dishes and cultured for 24 h in complete RPMI 1640. The medium was then changed to serum-free medium, and cells were further cultured for 24 h. Then, cells were treated with RES at 25 µM for 15 min. Cells were washed once with 1X PBS and fixed with 4% paraformaldehyde for 15 min. Sample coverslips were washed 3 times with PBS (5 min each time) on an orbital shaker and then permeabilized with 0.3% TritonX-100 in PBS for 5 min. After washing thrice, sample coverslips were blocked with 1% BSA in PBS for 1 h at RT and incubated with primary antibodies in a moist chamber at 4 °C overnight. Sample coverslips were washed 3 times with 1X PBS and incubated with a secondary antibody conjugated with Alexa488 or Alexa594 for 2 h in a moist chamber at RT. Sample coverslips were washed 3 times with 1X PBS and 1 time with deionized water (5 min) before mounting with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Signals of the target proteins were visualized at 100× magnification using a fluorescent microscope, Axio Vert.A1 (Carl Zeiss Suzhou Co., Ltd., Suzhou, China). The Zen version 2.6 (blue edition) software for the Zeiss Axiocam 506 color microscope camera was used to capture and analyze the images.