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Chromosome Research

, Volume 20, Issue 7, pp 849–858 | Cite as

Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections

  • Anja Eberhart
  • Hiroshi Kimura
  • Heinrich Leonhardt
  • Boris Joffe
  • Irina Solovei
Article

Abstract

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.

Keywords

Nuclear architecture epigenetic histone marks nuclear proteins mouse tissues mouse retina cryosections immunostaining antigen retrieval tissue fixation 

Abbreviations

AR

Antigen retrieval

DAPI

4′,6-Diamidino-2-phenylindole

GCL

Ganglion cell layer

H3K4me2

Di-methylated lysine 4 of histone H3

H3K36me3

Tri-methylated lysine 36 of histone H3

H3K27me3

Tri-methylated lysine 27 of histone H3

H4K8ac

Acetylated lysine 8 of histone H4

H4K20me2

Di-methylated lysine 20 of histone H4

HP1

Heterochromatin protein 1

INL

Inner nuclear layer

ONL

Outer nuclear layer

PBS

Phosphate-buffered saline

RNA Pol-II CTD

Non-phosphorilated carboxy-terminal domain of RNA polymerase II

RNA Pol-II Ser2ph

Phosphorylated serine 2 of heptapeptide repeat on carboxy-terminal domain of RNA polymerase II

RT

Room temperature

Notes

Acknowledgments

This research is supported by the German Research Foundation (DFG) SO1054/1 to IS, JO903/1 to BJ, SFB/TR5 to HL, and by grants-in-aid from the MEXT of Japan to HK.

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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Anja Eberhart
    • 1
  • Hiroshi Kimura
    • 2
  • Heinrich Leonhardt
    • 1
  • Boris Joffe
    • 1
  • Irina Solovei
    • 1
  1. 1.Department of Biology IIBiocenter Ludwig-Maximilians University MunichPlanegg-MartinsriedGermany
  2. 2.Graduate School of Frontier BiosciencesOsaka UniversitySuitaJapan

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