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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Nat Methods. 2013 Jan 13;10(2):171–177. doi: 10.1038/nmeth.2332

Figure 1. ISH-PLA: a new method of detection of histone modificationsat a single genomic locus in tissuesections.

Figure 1

(a) Schematic of the combined in situ hybridization (ISH) and Proximity Ligation Assay (PLA) method for detecting H3K4dime of the MYH11 promoter. (b) Workflow of the ISH-PLA procedures. (c) Immunostaining of 5 μm-thick sections of human carotid artery for ACTA2 and DAPI (diamidinophenyindole). Two distinct layers are identified: the media (M) and the adventitia (A). Small vessels consisting of ACTA2+ SMCs are visualized within the adventitia (arrows). Scale bar = 100 μm. (d) ISH-PLA in adventitial small arteries (n = 5). PLA amplification (PLA+) is visualized as a red spot localized within nuclei. MYH11 H3K4dime PLA+ signal is restricted to ACTA2+ medial SMCs (arrows) and is absent from CD31+ ECs (stars) as well as adventitial fibroblasts (arrow heads). (i–iii): High magnification with i) ACTA2, ii) PLA and iii) merge.Scale bar = 50 μm. (e)ISH-PLA negative control using an empty vector probe in adventitial vessel of human carotid artery sections. A total absence of PLA amplification demonstrates that hybridization of the biotin-labeled MYH11 probe is required for ISH-PLA amplification. Scale bar = 50 μm (f) Conventional ChIP assays showing enrichment of H3K4dime of MYH11 in SMCs but not in ECs. Mean ± s.d.; n = 3; *P < 0.05. (g) ISH-PLA in SMCs (n = 3) and ECs (n = 3) in vitro. MYH11 H3K4dime PLA amplification is restricted to SMCs.Scale bars = 10 μm.