title: Brilliant Blue G double staining enhances successful internal limiting membrane peeling with minimal adverse effect by low cellular permeability into live cells.
abstract: OBJECTIVE
  Brilliant Blue G is used as a surgical adjuvant for retinal surgery. Although BBG double or multiple staining was reported, the effectiveness and safety of repeated staining is still elusive. To further examine the effectiveness and safety, we examined BBG in clinical cases in vivo, primary cell culture in vitro, and surgically resected specimen ex vivo.
  
  
  METHODS
  A retrospective interventional case series with in vitro and ex vivo studies were performed. Vitrectomy was performed in 28 cases of epiretinal membrane with BBG single to multiple staining. The surgically resected membranes were stained by BBG with or without cellular fixation. Primary cell cultures were examined with BBG and live/death cell markers, such as Calcein AM and TUNEL.
  
  
  RESULTS
  Single staining provided satisfactory staining in seven cases. Double or multiple staining substantially visualized internal limiting membrane (21 cases), especially the edges of remaining internal limiting membrane (11 cases). Adverse retinal staining was not noted and the final visual acuity showed no difference with multiple staining. The live cells barely stained with BBG, while some dead cells were stained.
  
  
  CONCLUSIONS
  Brilliant Blue G multiple staining substantially enhanced the visualization of internal limiting membrane. The absence of abnormal staining supports the safety of repeated BBG staining.
authors.affiliation: *Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; †Clinical Research Institute, Kyushu Medical Center, Fukuoka, Japan; and ‡Department of Ophthalmology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Hisatomi
authors.forename: Shoji
authors.initials: S
authors.lastname: Notomi
authors.forename: Takashi
authors.initials: T
authors.lastname: Tachibana
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Oishi
authors.forename: Ryo
authors.initials: R
authors.lastname: Asato
authors.forename: Takehiro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Murakami
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Enaida
authors.forename: Taiji
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Ishibashi
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000378:metabolism / Q000473:pathology*; D002470:Cell Survival; D002478:Cells, Cultured; D019773:Epiretinal Membrane / Q000175:diagnosis / Q000378:metabolism / Q000601:surgery*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D007202:Indicators and Reagents; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010539:Permeability; D058486:Receptors, Purinergic P2X7 / Q000502:physiology; D012160:Retina / Q000166:cytology / Q000378:metabolism; D012189:Retrospective Studies; D012394:Rosaniline Dyes; D013194:Staining and Labeling; D041623:Tomography, Optical Coherence; D014821:Vitrectomy
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C546469:P2rx7 protein, mouse; D058486:Receptors, Purinergic P2X7; D012394:Rosaniline Dyes; C085925:calcein AM; C004692:coomassie Brilliant Blue