Abstract
The localization of nuclear proteins and modified histone tails changes during cell differentiation at the tissue as well as at the cellular level. Immunostaining in paraffin sections is the most powerful approach available to evaluate protein localization. Since nuclear proteins are sensitive to fixation, immunohistochemical conditions should be optimized in light of the particular antibodies and tissues employed. In this study, we searched for optimal conditions to detect histone modification at histone H3 lysine 9 (H3K9) and H3K9 methyltransferase G9a in the testis and cartilage in paraffin sections. In the testis, antigen retrieval (AR) was indispensable for detecting H3K9me1 and me3, G9a, and nuclear protein proliferating cell nuclear antigen (PCNA). With AR, shorter fixation times yielded better results for the detection of G9a and PCNA. Without AR, H3K9me2 and H3K9ac could be detected at shorter fixation times in primary spermatocytes of the testis. In contrast to the testis, all antibodies tested could detect their epitopes irrespective of AR application in the growth plate cartilage. Thus, conditions for the detection of epigenetic marks and nuclear proteins should be optimized in consideration of fixation time and AR application in different tissues and antibodies.
Highlights
Tissue-specific factors are expressed exclusively in certain groups of cells during cellular differentiation and cell fate determination
Since nuclear proteins are primarily localized in the nucleus, where DAPI staining labels DNA, the results are displayed in three panels: DAPI-stained, antibody-stained, and merged (Figures 1 and 3)
In the absence of antigen retrieval (AR) application, signals corresponding to H3K9me1, H3K9me3, G9a, and proliferating cell nuclear antigen (PCNA) were not detected in any nuclei in the spermatogonia and primary spermatocytes of the testis
Summary
Tissue-specific factors are expressed exclusively in certain groups of cells during cellular differentiation and cell fate determination. The transcriptionally competent state is characterized by an open chromatin locus, which is accessible to tissue-specific factors. Open or closed chromatin structures are characterized by the acetylation or methylation of histone tails, which are referred to as epigenetic marks, as well as chromatin-modifying nuclear proteins [4,5,6]. There are four methylated states at lysine 9 of histone H3: non-, mono-, di-, and trimethylated H3K9. These methylated states are determined by the balance of methyltransferases and demethylases. Elucidation of the localization and abundance of epigenetic marks and chromatin-modifying factors is essential for understanding the epigenetic regulation of cellular differentiation
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