Staining Protocols for Parasitic Worm Analysis

For Fast Blue B staining, female worms were fixed in 70% ethanol for ≥24 h. After staining with filtered 1% (w/v) Fast Blue B solution, they were dehydrated through an ethanol gradient from 70 to 100%, and then mounted in neutral balsam (Sinopharm Chemical Reagent Co., Ltd, China)66 (link). Staining of vitelline cells was evaluated using light microscopy (Nikon NI-SS, Japan). For hydrochloric carmine staining, worms were separated by sex and fixed in AFA (alcohol 95%, formalin 3%, glacial acetic acid 2%). Worms were stained with hydrochloric carmine for 30 min and destained in acidic 70% ethanol. After sequential dehydration in graded ethanol (70, 90, 100%), worms were mounted on glass slides with neutral balsam. Confocal images were taken with a Leica TCS-SP5 Spectral Laser Scanning Confocal Microscope (Leica, Germany) using a 488-nm He/Ne laser.

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Wang J., Yu Y., Shen H., Qing T., Zheng Y., Li Q., Mo X., Wang S., Li N., Chai R., Xu B., Liu M., Brindley P.J., McManus D.P., Feng Z., Shi L, & Hu W. (2017). Dynamic transcriptomes identify biogenic amines and insect-like hormonal regulation for mediating reproduction in Schistosoma japonicum. Nature Communications, 8, 14693.

Publication 2017

neutral balsam NI-SS Leica TCS-SP5 Spectral Laser Scanning Confocal Microscope

Acidic Alcohol Carmine Cells Confocal laser scanning microscope Dehydration Ethanol Fast blue b Female Formalin Glacial acetic acid Light microscopy Worms

Corresponding Organization :

Other organizations : Fudan University, National Institute for Parasitic Diseases, George Washington University, QIMR Berghofer Medical Research Institute

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Variable analysis

independent variables

Staining method (Fast Blue B, hydrochloric carmine)

dependent variables

Staining of vitelline cells
Confocal images

control variables

Fixation of female worms in 70% ethanol for ≥24 h
Dehydration of worms through an ethanol gradient from 70 to 100%
Mounting of worms in neutral balsam
Separation of worms by sex (for hydrochloric carmine staining)
Fixation of worms in AFA (alcohol 95%, formalin 3%, glacial acetic acid 2%)
Staining with hydrochloric carmine for 30 min
Destaining in acidic 70% ethanol
Sequential dehydration in graded ethanol (70, 90, 100%)
Mounting of worms on glass slides with neutral balsam
Use of Leica TCS-SP5 Spectral Laser Scanning Confocal Microscope with 488-nm He/Ne laser

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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