Japanese Journal of Ophthalmology

, Volume 62, Issue 4, pp 481–490 | Cite as

Optimization of fixative solution for retinal morphology: a comparison with Davidson’s fixative and other fixation solutions

  • Kazuhiro TokudaEmail author
  • Byron Baron
  • Yasuhiro Kuramitsu
  • Takao Kitagawa
  • Nobuko Tokuda
  • Naoyuki Morishige
  • Masaaki Kobayashi
  • Kazuhiro Kimura
  • Kazuyuki Nakamura
  • Koh-Hei Sonoda
Laboratory Investigation



Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson’s fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions.

Study design

Experimental design.


Four fixative formulations (4% paraformaldehyde, Davidson’s fixative, modified Davidson’s fixative and an in-house fixative – TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared.


TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Müller cells), when compared to FA, Davidson’s fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily.


It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.


Retinal fixation Crosslinking fixative Coagulative fixative Hematoxylin and eosin staining Immunohistochemistry 



The authors thank Junzo Kitoh for critical advice. The authors thank Yukari Mizuno, Shizuka Murata, Fusako Itoh and Mika Tominaga for technical assistance and support. This work was supported by JSPS KAKENHI Grant number JP16K11323.

Conflicts of interest

K. Tokuda, None; B. Baron, None; Y. Kuramitsu, None; T. Kitagawa, None; N. Tokuda, None; N. Morishige, None; M. Kobayashi, None; K. Kimura, None; K. Nakamura, None; K. Sonoda, None.

Supplementary material

10384_2018_592_MOESM1_ESM.pdf (205 kb)
Supplementary material 1 (PDF 205 kb)


  1. 1.
    Schlosshauer B, Grauer D, Dütting D, Vanselow J. Expression of a novel Müller glia specific antigen during development and after optic nerve lesion. Development. 1991;111:789–99.PubMedGoogle Scholar
  2. 2.
    Shinoe T, Kuribayashi H, Saya H, Seiki M, Aburatani H, Watanabe S. Identification of CD44 as a cell surface marker for Müller glia precursor cells. J Neurochem. 2010;115:1633–42.CrossRefPubMedGoogle Scholar
  3. 3.
    Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory and practice of fixation of tissues. J Histotechnol. 2001;24:173–90.CrossRefGoogle Scholar
  4. 4.
    Eldred WD, Zucker C, Karten HJ, Yazulla S. Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry. J Histochem Cytochem. 1983;31:285–92.CrossRefPubMedGoogle Scholar
  5. 5.
    Izumi Y, Hammerman SB, Benz AM, Labruyere J, Zorumski CF, Olney JW. Comparison of rat retinal fixation techniques: chemical fixation and microwave irradiation. Exp Eye Res. 2000;70:191–8.CrossRefPubMedGoogle Scholar
  6. 6.
    McMenamin PG. Optimal methods for preparation and immunostaining of iris, ciliary body, and choroidal wholemounts. Investig Ophthalmol Vis Sci. 2000;41:3043–8.Google Scholar
  7. 7.
    Grizzle WE, Fredenburgh J, Myers RB. Fixation of tissues. In: Bancroft J, Gamble M, editors. Theory and practice of histological techniques. 6th ed. Edinburgh: Churchill Livingstone; 2007. p. 53–74.Google Scholar
  8. 8.
    McKay JS, Steele SJ, Ahmed G, Johnson E, Ratcliffe K. An antibody panel for immunohistochemical analysis of the retina in Davidson’s-fixed, paraffin-embedded eyes of rats. Exp Toxicol Pathol. 2009;61:91–100.CrossRefPubMedGoogle Scholar
  9. 9.
    Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM. Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol Pathol. 2002;30:524–33.CrossRefPubMedGoogle Scholar
  10. 10.
    Chidlow G, Daymon M, Wood JP, Casson RJ. Localization of a wideranging panel of antigens in the rat retina by immunohistochemistry: comparison of Davidson’s solution and formalin as fixatives. J Histochem Cytochem. 2011;59:884–98.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tokuda K, Zorumski CF, Izumi Y. Involvement of illumination in indocyanine green toxicity after its washout in the ex vivo rat retina. Retina. 2009;29:371–9.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ogata G, Stradleigh TW, Partida GJ, Ishida AT. Dopamine and full-field illumination activate D1 and D2–D5-type receptors in adult rat retinal ganglion cells. J Comp Neurol. 2012;520:4032–49.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Margo CE, Lee A. Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact. Graefes Arch Clin Exp Ophthalmol. 1995;233:366–70.CrossRefPubMedGoogle Scholar
  14. 14.
    Vinores SA, Derevjanik NL, Mahlow J, Hackett SF, Haller JA, deJuan E, et al. Class III beta-tubulin in human retinal pigment epithelial cells in culture and in epiretinal membranes. Exp Eye Res. 1995;60:385–400.CrossRefPubMedGoogle Scholar
  15. 15.
    Powner MB, Gillies MC, Zhu M, Vevis K, Hunyor AP, Fruttiger M. Loss of Müller’s cells and photoreceptors in macular telangiectasia type 2. Ophthalmology. 2013;120:2344–52.CrossRefPubMedGoogle Scholar
  16. 16.
    Vardimon L, Fox LE, Moscona AA. Developmental regulation of glutamine synthetase and carbonic anhydrase II in neural retina. Proc Natl Acad Sci USA. 1986;83:9060–4.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kondo H, Iwanaga T, Nakajima T. An immunocytochemical study on the localization of S-100 protein in the retina of rats. Cell Tissue Res. 1983;231:527–32.CrossRefPubMedGoogle Scholar
  18. 18.
    Dyer MA, Cepko CL. Control of Müller glial cell proliferation and activation following retinal injury. Nat Neurosci. 2000;3:873–80.CrossRefPubMedGoogle Scholar
  19. 19.
    Thomas P, Smart TG. HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods. 2005;51:187–200.CrossRefPubMedGoogle Scholar
  20. 20.
    Heegaard S, Rosenberg T, Preising M, Prause JU, Bek T. An unusual retinal vascular morphology in connection with a novel AIPL1 mutation in Leber’s congenital amaurosis. Br J Ophthalmol. 2003;87:980–3.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zhong X, Ge J, Smith EL 3rd, Stell WK. Image defocus modulates activity of bipolar and amacrine cells in macaque retina. Investig Ophthalmol Vis Sci. 2004;45:2065–74.CrossRefGoogle Scholar
  22. 22.
    Tabandeh H, Dubovy S, Green WR. Bilateral midperipheral large drusen and retinal pigment epithelial detachments associated with multifocal areas of choroidal neovascularization: a histopathologic study. Retina. 2006;26:1063–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Bell WR, Green WR, Goldberg MF. Histopathologic and trypsin digestion studies of the retina in incontinentia pigmenti. Ophthalmology. 2008;115:893–7.CrossRefPubMedGoogle Scholar
  24. 24.
    Stradleigh TW, Ishida AT. Fixation strategies for retinal immunohistochemistry. Prog Retin Eye Res. 2015;48:181–202.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kiernan JA. Histological and histochemical methods: theory and practice. 5th ed. Banbury: Scion Publishing; 2015.Google Scholar
  26. 26.
    Helander KG. Kinetic studies of formaldehyde binding in tissue. Biotech Histochem. 1994;69:177–9.CrossRefPubMedGoogle Scholar
  27. 27.
    Ghoufi A, Artzner F, Malfreyt P. Physical properties and hydrogen-bonding network of water–ethanol mixtures from molecular dynamics simulations. J Phys Chem B. 2016;120:793–802.CrossRefPubMedGoogle Scholar
  28. 28.
    Tzekov R, Lin T, Zhang KM, Jackson B, Oyejide A, Orilla W, et al. Ocular changes after photodynamic therapy. Investig Ophthalmol Vis Sci. 2006;47:377–85.CrossRefGoogle Scholar
  29. 29.
    Marangoni D, Bush RA, Zeng Y, Wei LL, Ziccardi L, Vijayasarathy C, et al. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice. Mol Ther Methods Clin Dev. 2016;5:16011.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Steinberg RH, Reid M, Lacy PL. The distribution of rods and cones in the retina of the cat (Felis domesticus). J Comp Neurol. 1973;148:229–48.CrossRefPubMedGoogle Scholar
  31. 31.
    Pow DV, Wright LL, Vaney DI. The immunocytochemical detection of amino-acid neurotransmitters in paraformaldehyde-fixed tissues. J Neurosci Methods. 1995;56:115–23.CrossRefPubMedGoogle Scholar
  32. 32.
    Berod A, Hartman BK, Pujol JF. Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine-hydroxylase. J Histochem Cytochem. 1981;29:844–50.CrossRefPubMedGoogle Scholar
  33. 33.
    Moran J, Hurtado S, Pasantes-Morales H. Similar properties of taurine release induced by potassium and hyposmolarity in the rat retina. Exp Eye Res. 1991;53:347–52.CrossRefPubMedGoogle Scholar
  34. 34.
    Villegas GM. Ultrastructure of the human retina. J Anat. 1964;98:501–13.PubMedPubMedCentralGoogle Scholar

Copyright information

© Japanese Ophthalmological Society 2018

Authors and Affiliations

  • Kazuhiro Tokuda
    • 1
    • 2
    Email author
  • Byron Baron
    • 2
    • 3
  • Yasuhiro Kuramitsu
    • 2
    • 4
  • Takao Kitagawa
    • 2
  • Nobuko Tokuda
    • 5
  • Naoyuki Morishige
    • 1
  • Masaaki Kobayashi
    • 1
  • Kazuhiro Kimura
    • 1
  • Kazuyuki Nakamura
    • 2
  • Koh-Hei Sonoda
    • 6
  1. 1.Department of OphthalmologyYamaguchi University Graduate School of MedicineUbeJapan
  2. 2.Department of Systems Biochemistry in Pathology and RegenerationYamaguchi University Graduate School of MedicineUbeJapan
  3. 3.Faculty of Medicine and Surgery, Centre for Molecular Medicine and BiobankingUniversity of MaltaMsidaMalta
  4. 4.Research Institute of Cancer PreventionHealth Sciences University of HokkaidoHokkaidoJapan
  5. 5.Faculty of Health SciencesYamaguchi University Graduate School of MedicineUbeJapan
  6. 6.Department of Ophthalmology, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan

Personalised recommendations