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. 1. Introduction Tissue-specific factors are expressed exclusively in certain groups of cells during cellular differentiation and cell fate determination. Genes activated during differentiation are maintained in a transcriptionally competent state in chromatin, whereas genes that are not activated in a given lineage are maintained in a silenced state. The transcriptionally competent state is characterized by an open chromatin locus, which is accessible to tissue-specific factors. In a silenced state, transcriptionally inactive condensed chromatin is formed [1–3]. 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 [4–6]. The modifications of histone tails are regulated by histone modification enzymes. For example, 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. The key histone methyltransferases is G9a, which is a member of the Suv39h subgroup of SET domain-containing molecules [7]. G9a is responsible for the modification of H3K9me1 and H3K9me2, and affects chromatin status, leading to gene
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