Journal: Molecular and cellular neurosciences
Article Title: Hippocampal Stimulation Promotes Intracellular Tip60 Dynamics with Concomitant Genome Reorganization and Synaptic Gene Activation.
doi: 10.1016/j.mcn.2019.103412
Figure Lengend Snippet: (A) Representative images of quadruple-label immuno-DNA FISH of DIV 12 rat hippocampal neurons under unstimulated and KCl stimulated conditions. Immunofluorescence detection of TFs by RNAPII S2P staining (red), Tip60 foci (yellow), gene loci detected by custom single-stranded FISH probes (green) and nuclei counterstained with DAPI (blue). Enlarged images show selected gene loci (green, outlined in white). Scale bar indicates 1 μm. (B) The histogram represents the percentage of genes within TFs found associated with Tip60 in hippocampal neurons under unstimulated and stimulated conditions as determined by quadruple-label immuno-DNA FISH. Of note, and similar to prior publications (CREPALDI et al, 2013), in many representative images, one allele is observed as the other allele is often located within a different Z stack. Thus, multiple FISH signals per gene loci per condition are assessed. Data are represented as the percentage of colocalization [(number of positive cells/total number of cells counted)*100]. A Fisher’s Exact test was conducted to measure significance in the association between colocalization of gene loci, TFs and Tip60, and stimulation condition. Here, n = 8 to 13 FISH signals per gene loci per condition. See Supplementary Table 4 for details on analysis.
Article Snippet: Experimental Model and Subject Details Source of cell lines used in this study is reported in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Tip60 Santa Cruz Biotechnology Cat#166323; RRID: AB_2296327 Rabbit polyclonal anti-Tip60 Abcam Cat# ab23886; RRID: AB_778485 Mouse monoclonal anti-MAP-2 Santa Cruz Biotechnology Cat#sc-74421; RRID: AB_1126215 Rabbit polyclonal anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) Abcam Cat# ab5095; RRID: AB_304749 Goat polyclonal anti-Rabbit IgG, Alexa Fluor 647 Jackson ImmunoResearch Labs Cat# 111–607003; RRID: AB_2338084 Goat anti-Mouse IgG, Cy3 Abcam Cat# ab97035; RRID: AB_10680176 Chemicals, Peptides, And Recombinant Proteins Prolong Gold Antifade w/DAPI Molecular Probes {"type":"entrez-protein","attrs":{"text":"P36941","term_id":"549090","term_text":"P36941"}} P36941 NHS-Fluorescein (5/6-carboxyfluorescein succinimidyl ester), mixed isomer Thermo Fisher 46410 Streptavidin-Alexa568 Thermo Fisher {"type":"entrez-protein","attrs":{"text":"S11226","term_id":"110724","term_text":"pir||S11226"}} S11226 AminoAllyl-dUTP Thermo Fisher FERR0091 Biotin-11-dUTP Thermo Fisher R0081 Critical Commercial Assays EZ-Magna ChIP A Kit EZ-Magna ChIP A Kit EZ-Magna ChIP A Kit TruChIP Chromatin Shearing Kit TruChIP Chromatin Shearing Kit TruChIP Chromatin Shearing Kit Taq DNA Polymerase Kit Taq DNA Polymerase Kit Taq DNA Polymerase Kit Blood & Cell Culture DNA Mini Kit Blood & Cell Culture DNA Mini Kit Blood & Cell Culture DNA Mini Kit NucleoSpin Gel and PCR Clean-Up (50) NucleoSpin Gel and PCR Clean-Up (50) NucleoSpin Gel and PCR Clean-Up (50) RNeasy Mini Kit RNeasy Mini Kit RNeasy Mini Kit Superscript II Reverse Transcriptase Kit Invitrogen 18064022 Experimental Models: Cell Lines Rattus Norvegicus Hippocampal Cultures University of Pennsylvania, Mahoney Institute for Nueroscience Neuron Culture Service Center http://www.med.upenn.edu/neuronsrus Oligonucleotides Primers for RT-qPCR, see Table S1 Primers for ChIP, see Table S1 Primers for FISH Probe Generation, see Table S1 Software and Algorithms ImageJ NIH https://imagej.net Olympus fluoview acquisition software Olympus, Center Valley, PA IBM SPSS Statistics IBM Analytics https://www.ibm.com/analytics/data-science/predictive-analytics/spss-statistical-software Corrected Total Cellular Fluorescence (CTCF) McCloy, 2014 ; Gavet, 2010 http://doi.org.ezproxy2.library.drexel.edu/10.4161/cc.28401 . http://doi.org.ezproxy2.library.drexel.edU/10.1016/j.devcel.2010.02.013 Open in a separate window KEY RESOURCES TABLE
Techniques: Immunofluorescence, Staining
Santa Cruz Biotechnology is a verified supplier 
![(A) Representative images of quadruple-label immuno-DNA FISH of DIV 12 rat hippocampal neurons under unstimulated and KCl stimulated conditions. Immunofluorescence detection of TFs by RNAPII S2P staining (red), Tip60 foci (yellow), gene loci detected by custom single-stranded FISH probes (green) and nuclei counterstained with DAPI (blue). Enlarged images show selected gene loci (green, outlined in white). Scale bar indicates 1 μm. (B) The histogram represents the percentage of genes within TFs found associated with Tip60 in hippocampal neurons under unstimulated and stimulated conditions as determined by quadruple-label immuno-DNA FISH. Of note, and similar to prior publications (CREPALDI et al, 2013), in many representative images, one allele is observed as the other allele is often located within a different Z stack. Thus, multiple FISH signals per gene loci per condition are assessed. Data are represented as the percentage of colocalization [(number of positive cells/total number of cells counted)*100]. A Fisher’s Exact test was conducted to measure significance in the association between colocalization of gene loci, TFs and Tip60, and stimulation condition. Here, n = 8 to 13 FISH signals per gene loci per condition. See Supplementary Table 4 for details on analysis.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9871/pmc06879871/pmc06879871__nihms-1542566-f0005.jpg)
