Files/images of the individual channels of the composite images used as an example are included as Supplement Files 2, 3, 4 [which are images of immunofluorescent staining with Hoechst (blue), Lac-I (green), and Brm (red, originally described in Nakayama (2019). approach to assess chromatin changes at genomic sites, ectopically targeted by nuclear pore proteins. The use of our analysis program increases the ability to make unbiased conclusions on changes in chromatin structure, or in protein recruitment to chromatin, regardless of sample variation AC-55541 in immunofluorescence staining. As it is simply based upon differences in fluorescence intensity at a defined location, the provided analysis program is not limited to analysis of polytene chromosome, and could be applied to many different contexts where correlation between fluorescent signals at any particular location is of interest. larval salivary glands have an unusually large size (visible under a typical cell-culture microscope). This is due to altered cell cycle processes that result in ~1,000 copies of each chromosome that do not segregate from each other. The resulting large chromosomes have stereotyped and reproducible alternating bands of condensed and decondensed chromatin, as chromosomes are aligned and locus-specific chromatin structure is visible upon polytenization. The nature of these chromosomes makes the analysis of chromatin structure uniquely visible relative to diploid cell types, enabling analysis of chromatin changes throughout development, transcriptional activation, or upon perturbation. Additionally, genome-wide and site-specific chromatin binding of proteins can be visualized by immunofluorescence staining of polytene chromosomes (Paro, 2008; Cai repeat array ( Kuhn arrays, integrated at defined sites in the genome. We then aimed to determine whether such targeting leads to recruitment of candidate interacting partners, and also to changes in target chromatin compaction. This approach involved identification of a single band of interest (harboring the site) within an entire genome of spread chromosomes, which required high-quality chromosome spreading for increased visibility and analysis of chromosome banding patterns. Additionally, screening through multiple antibodies to assay for many candidate interacting partners required an extremely reproducible squashing protocol, with minimal sample failure rate. To achieve these goals, we developed this optimized polytene squashing protocol, which can be utilized to improve image quality and sample success rate. It is also especially useful for chromosome mappability (determining genomic location by cytological mapping of polytene banding patterns) and for more high-throughput applications. In order to reliably analyze site-specific changes in chromatin structure (larvae, there are a number of public videos available, available here) Peel off the fat bodies (white strips of tissue) from the sides of glands, as this can contribute to fluorescence background in downstream imaging; however dont spend more than a couple of minutes removing these if they are persistent, it is better to proceed to fixation sooner. Dissections should not proceed longer than ~5 min before fixation. Transfer the glands to a different dissection dish well made up of ~100-200 l of freshly made fixation answer and leave for precisely 1 min. Glands may be held by the wishbone/stem area where they are adjoined to transfer to fixative answer, as this prevents damaging Rabbit Polyclonal to CDK5 gland cells. AC-55541 Transferring glands here can be made much easier by the use of a pair of forceps with one bent/curved tip, described in Step B7 below. Acetic acid serves here to AC-55541 disrupt nuclear membranes and AC-55541 aid in chromosome spreading, however not all antibodies work well under these conditions, and AC-55541 some may require optimization in different fixation lengths or solutions, see Note in Section C. staining process, from hammering to final coverslip mounting. Scale bar = 10 m. C. Well-spread, but under-flattened chromosomes, evident by chromosomes present in multiple focal planes, integration site at cytological site.