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

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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.

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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

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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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Authors and Affiliations

  1. Department of Biology II, Biocenter Ludwig-Maximilians University Munich, Grosshadernerstrasse 2, 82152, Planegg-Martinsried, Germany

    Anja Eberhart, Heinrich Leonhardt, Boris Joffe & Irina Solovei

  2. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Hiroshi Kimura

Authors
  1. Anja Eberhart
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  2. Hiroshi Kimura
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  3. Heinrich Leonhardt
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  4. Boris Joffe
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  5. Irina Solovei
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Corresponding author

Correspondence to Irina Solovei.

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Responsible Editor: Conly Rieder.

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Eberhart, A., Kimura, H., Leonhardt, H. et al. Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections. Chromosome Res 20, 849–858 (2012). https://doi.org/10.1007/s10577-012-9318-8

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  • DOI: https://doi.org/10.1007/s10577-012-9318-8

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