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| '''Papers and other sites:'''
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| *Davidson, L. A., and Keller, R. E. (1999). Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation, and convergent extension. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10498689| Development, 126:4547-4556]
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| *Zhou, X. and Vize, P.D. (2004). Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric tubules. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15223337| Developmental Biology 271: 322-338.]
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| *Davidson, Keller, and DeSimone (2004) Patterning and tissue movements in a novel explant preparation of the marginal zone of Xenopus laevis. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15183313| Gene Expression Patterns Jul;4(4):457-66.]
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| *[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html| Lance Davidson's flourescent in situ methods page]
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| '''Protocol'''
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| The generation of fixable fluorescent products relies on a tyramide substrate. This is literally a tyramide linked to your favorite fluor. The tyramide is converted to a reactive form by HRP in the presence of H2O2, and this reactive form binds to a local protein (usually the ring on tyrosines...), thus localizing and fixing your fluor. Following the reaction you wash out the unreacted tyramide conjugant and visualize using fluorescence or confocal microscopy.
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| You can purchase tyramides conjugated to various fluors from Molecular Probes, Perkin Elmer etc. We have tried some of these and NONE worked- the Molecular Probes kits gave zero signal. It is trivial to make your own, as outlined in the linked pages. The only issue here is where you get your NHS-fluors, once again some we have purchased did not work at all. A listing of the good, bad and ugly appears below. To date we have had the best luck with [[Flourescin Tyramide Synthesis| NHS-FITC]] from Pierce and [[Cy3 Tyramide Synthesis| NHS-Cy3 from Amersham]].
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| The fluor is stable to embryo bleaching in standard methanol/peroxide bleach, so you can stain then bleach as per colorimetric in situs, at least for [[Flourescin Tyramide Synthesis| FITC]] and [[Cy3 Tyramide Synthesis| Cy3]]. FITC fades a little on bleaching, Cy3 is rock solid.
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| We often use fluorescent in situ as a counterstain to normal BM-purple in situs. You can overlay the images and get effective overlaps, or simply use the fluor for contextual detail visually. Details of the combination, and the Photoshop tricks used to generate images such as the one above, are from the Dev Biol. paper, but are also outlined on the [[FCIS| FCIS page]].
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| Perform in situ hybridization using the standard protocol from the CSH manual or the [http://tropicalis.berkeley.edu/home/gene_expression/in-situ/insitu.html| Harland lab]. Use DIG, FITC or DNP labeled probes. Detect bound probe using a HRP-coupled antibody. If you are using a DIG labeled antisense mRNA use:
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| Roche Anti-DIG-POD (POD = HRP) cat # 1 207 733
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| If you have a FITC labelled RNA use:
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| Roche anti-fluorescein-POD cat# 1 426 346
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| The following is all performed in stardard 5ml glass vials
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| # following in situ protocol wash in PBST (PBS with 0.01% Tween 20).
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| # add H2O2 to final conc. of 2%. incubate 60 minutes at room temp.
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| # wash twice with TBST (12 mMTris pH7,5, 150 mM NaCl, 0.01% Tween20)
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| # add standard antibody blocking solution, after 5 minutes add Roche Anti-DIG-POD antibody at 1:1000. incubate at 4 deg C overnight with rocking.
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| # wash 5 x 1hr at room temp in TBST
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| # wash in PBST 2 x 10 min. This is important- do not use TBS. add tyramide-FITC at 1:100 to 1:1,000
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| # incubate at room temp. for 20 minutes
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| # add H2O2 to final conc. of 0.001% (2 ul of 1/1000 diluted stock to 200ul PBST)
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| # incubate at room temp for 30 - 40 minutes
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| # for single color wash in TBST for 24 - 48 hours (or more) with multiple changes. (Yes, one to 2 days!). For two or three color go onto next step (wash out all colors together later)
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| # to do a second color begin again at step 2, the high H2O2 will inactivate the previously bound HRP, then skip to step 6. 2% H2O2 should stop any cross-over, but 3% is also fine if you have any trouble.
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| # wash for one to 2 days with rocking. We usually wash at 4 deg C over the weekend.
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| Weak signals? Lance recommends doing repeated tyramide cycles, each one for 30 minutes. We find that simply increasing tyramide concentrations is even better and do just one cycle. this is why we use 1:100 tyramide or even 1:50, rather than the 1:1000 in other protocols. When you make it yourself its cheap and doesn't cause background.
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| '''Note:''' for double fluor reactions ...
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| Develop the Cy3 fluorescence first using a DIG labeled probe (you don't want the FITC-tyramide being detected by your anti-FITC antibody in round 2...).
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| DNP nucleotides and anti-DNP-HRP are available from Perkin-Elmer to use this option. it is more sensitive than FITC nucletides/antibodies from Roche.
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