Wholemount embryo FISH / Double FISH - Revision history

205.142.197.33 at 18:12, 12 June 2012

2012-06-12T18:12:24Z

← Older revision		Revision as of 18:12, 12 June 2012
Line 8:		Line 8:

	*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\| Lance Davidson's flourescent in situ methods page]		*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\| Lance Davidson's flourescent in situ methods page]

			*Vize, P.D., McCoy, K.E., and Zhou, X. (2009). Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos. [http://www.ncbi.nlm.nih.gov/pubmed/19498377\| Nat Protoc. 4(6): 975-983.]

imported>Xenbase: moved FISH / Double FISH to Wholemount embryo FISH / Double FISH

2011-01-10T17:21:18Z

moved FISH / Double FISH to Wholemount embryo FISH / Double FISH

← Older revision	Revision as of 17:21, 10 January 2011
(No difference)

imported>Xenbase: Undo revision 40450 by 83.234.245.250 (talk)

2010-11-11T16:57:26Z

Undo revision 40450 by 83.234.245.250 (talk)

← Older revision		Revision as of 16:57, 11 November 2010
Line 1:		Line 1:
	~~<br /><br /><center><h1> [http~~:~~//trusted-pharmacy.org/ CLICK HERE TO ENTER DRUGSTORE! - </h1><br /></center>~~		'''Papers and other sites:'''
	~~<br />~~
	~~<br /><br />~~
	~~<br /><br />~~
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	~~<br /><br />~~
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	~~<br /><br />~~
	~~<br /><br />~~
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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]

	~~zyrtec symptom s~~		*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.]
	~~<br~~ />
	~~granby fire mckenna ortho~~		*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.]
	~~<br~~ />
	~~ortho executives paul rocca~~		*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\| Lance Davidson's flourescent in situ methods page]
	~~<br~~ />
	~~medical experts vioxx~~
	~~<br~~ />		'''Protocol'''
	~~rivotril side effects~~
	~~<br~~ />		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.
	~~cost of risperdal~~		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]].
	~~<br~~ />
	~~ibuprofen dosage for 10 lb baby~~		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.
	~~<br~~ />
	~~vacu vin inc~~
	~~<br~~ />		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]].
	~~generic hydrocodone without acetaminophen~~
	~~<br~~ />		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:
	~~buy vicodin without prescription membership~~		Roche Anti-DIG-POD (POD = HRP) cat # 1 207 733
	~~<br />~~
	~~sleeping aids ambien~~		If you have a FITC labelled RNA use:
	~~<br />~~		Roche anti-fluorescein-POD cat# 1 426 346
	~~poss stick wang alcohol cancer genetic~~
	~~<br />~~		The following is all performed in stardard 5ml glass vials
	~~fetal alcohol spectrum disorder journal articles~~		# following in situ protocol wash in PBST (PBS with 0.01% Tween 20).
	~~<br~~ />		# add H2O2 to final conc. of 2%. incubate 60 minutes at room temp.
	~~depakote pregnancy transition~~		# wash twice with TBST (12 mMTris pH7,5, 150 mM NaCl, 0.01% Tween20)
	~~<br />~~		# 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.
	~~neurontin~~ and ~~medical id braclets~~		# wash 5 x 1hr at room temp in TBST
	~~<br~~ />		# wash in PBST 2 x 10 min. This is important- do not use TBS. add tyramide-FITC at 1:100 to 1:1,000
	~~effexor and heart problems~~		# incubate at room temp. for 20 minutes
	~~<br~~ />		# add H2O2 to final conc. of 0.001% (2 ul of 1/1000 diluted stock to 200ul PBST)
	~~meridia doamine~~		# incubate at room temp for 30 - 40 minutes
	~~<br~~ />		# 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)
	~~marijuana theme t~~-~~shirts~~		# 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.
	~~<br />~~		# wash for one to 2 days with rocking. We usually wash at 4 deg C over the weekend.
	~~oxycontin abuse facts~~
	~~<br />~~
	~~gold slogger alcohol~~		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.
	~~<br />~~
	~~ibuprofen neonates~~		'''Note:''' for double fluor reactions ...
	~~<br />~~
	~~remeron histamine~~
	~~<br />~~		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...).
	~~casino chips fort worth~~
	~~<br />~~		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.
	~~side effects~~ of ~~vicodin lortab~~
	~~<br~~ />
	~~congressman david loebsack~~
	~~<br />~~
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	~~vagov~~
	~~<br />~~
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imported>Xenbase: Reverted edits by Xenbase (talk) to last revision by 83.234.245.250

2010-11-11T16:56:54Z

Reverted edits by Xenbase (talk) to last revision by 83.234.245.250

← Older revision	Revision as of 16:56, 11 November 2010
Line 1:	Line 1:
		<br /><br /><center><h1> [http://trusted-pharmacy.org/ CLICK HERE TO ENTER DRUGSTORE! - </h1><br /></center>
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imported>Xenbase: Blanked the page

2010-11-11T16:54:43Z

Blanked the page

← Older revision		Revision as of 16:54, 11 November 2010
Line 1:		Line 1:
	~~<br /><br /><center><h1> [http://trusted-pharmacy.org/ CLICK HERE TO ENTER DRUGSTORE! - </h1><br /></center>~~
	~~<br />~~
	~~<br /><br />~~
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	~~depakote pregnancy transition~~
	~~<br />~~
	~~neurontin and medical id braclets~~
	~~<br />~~
	~~effexor and heart problems~~
	~~<br />~~
	~~meridia doamine~~
	~~<br />~~
	~~marijuana theme t-shirts~~
	~~<br />~~
	~~oxycontin abuse facts~~
	~~<br />~~
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	~~<br />~~
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	~~<br />~~
	~~remeron histamine~~
	~~<br />~~
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	~~<br />~~
	~~side effects of vicodin lortab~~
	~~<br />~~
	~~congressman david loebsack~~
	~~<br />~~
	~~discount airfare~~
	~~<br />~~
	~~weight gain with paxil~~
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83.234.245.250: CLICK HERE TO ENTER DRUGSTORE

2010-11-10T09:22:17Z

CLICK HERE TO ENTER DRUGSTORE

← Older revision		Revision as of 09:22, 10 November 2010
Line 1:		Line 1:
	~~'''Papers and other sites~~:~~'''~~		<br /><br /><center><h1> [http://trusted-pharmacy.org/ CLICK HERE TO ENTER DRUGSTORE! - </h1><br /></center>
			<br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />
			<br /><br />

	*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]

	*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.]~~		zyrtec symptom s
			<br />
	*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.]~~		granby fire mckenna ortho
			<br />
	*[http://~~www.faculty.virginia.edu/davidson/fluor_insitu~~/~~fluorescent_in_situ.html\| Lance Davidson's flourescent in situ methods page]~~		ortho executives paul rocca
			<br />
			medical experts vioxx
	~~'''Protocol'''~~		<br />
			rivotril side effects
	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.		<br />
	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]].~~		cost of risperdal
			<br />
	~~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.		ibuprofen dosage for 10 lb baby
			<br />
			vacu vin inc
	~~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]].		<br />
			generic hydrocodone without acetaminophen
	~~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:		<br />
	~~Roche Anti-DIG-POD (POD = HRP) cat # 1 207 733~~		buy vicodin without prescription membership
			<br />
	~~If you have a FITC labelled RNA use:~~		sleeping aids ambien
	~~Roche anti-fluorescein-POD cat# 1 426 346~~		<br />
			poss stick wang alcohol cancer genetic
	~~The following is all performed in stardard 5ml glass vials~~		<br />
	~~# following in situ protocol wash in PBST (PBS with 0.01% Tween 20).~~		fetal alcohol spectrum disorder journal articles
	~~# add H2O2 to final conc. of 2%. incubate 60 minutes at room temp.~~		<br />
	~~# wash twice with TBST (12 mMTris pH7,5, 150 mM NaCl, 0.01% Tween20)~~		depakote pregnancy transition
	~~# 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.~~		<br />
	~~# wash 5 x 1hr at room temp in TBST~~		neurontin and medical id braclets
	~~# wash in PBST 2 x 10 min. This is important- do not use TBS. add tyramide-FITC at 1:100 to 1:1,000~~		<br />
	~~# incubate at room temp. for 20 minutes~~		effexor and heart problems
	~~# add H2O2 to final conc. of 0.001% (2 ul of 1~~/~~1000 diluted stock to 200ul PBST)~~		<br />
	~~# incubate at room temp for 30 - 40 minutes~~		meridia doamine
	~~# 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)~~		<br />
	# 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.		marijuana theme t-shirts
	~~# wash for one to 2 days~~ with ~~rocking. We usually wash at 4 deg C over the weekend.~~		<br />
			oxycontin abuse facts
			<br />
	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.		gold slogger alcohol
			<br />
	~~'''Note:''' for double fluor reactions ...~~		ibuprofen neonates
			<br />
			remeron histamine
	~~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...).~~		<br />
			casino chips fort worth
	~~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.~~		<br />
			side effects of vicodin lortab
			<br />
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			<br />
			discount airfare
			<br />
			weight gain with paxil
			<br />
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			<br />
			vagov
			<br />
			cocaine in sears catalog
			<br />
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			<br />

136.159.4.28 at 20:54, 7 January 2010

2010-01-07T20:54:21Z

← Older revision		Revision as of 20:54, 7 January 2010
Line 7:		Line 7:
	*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.]		*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.]

	*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html \| Lance Davidson's flourescent in situ methods page]		*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\| Lance Davidson's flourescent in situ methods page]

136.159.4.28 at 20:54, 7 January 2010

2010-01-07T20:54:13Z

← Older revision		Revision as of 20:54, 7 January 2010
Line 5:		Line 5:
	*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.]		*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.]

	*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.]		*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.]

	*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\|Lance Davidson's flourescent in situ methods page]		*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html \| Lance Davidson's flourescent in situ methods page]

136.159.4.28 at 20:53, 7 January 2010

2010-01-07T20:53:59Z

← Older revision		Revision as of 20:53, 7 January 2010
Line 7:		Line 7:
	*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.]		*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.]

	*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\| Lance Davidson's flourescent in situ methods page]		*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\|Lance Davidson's flourescent in situ methods page]

imported>Xenbase at 18:59, 22 December 2009

2009-12-22T18:59:08Z

← Older revision		Revision as of 18:59, 22 December 2009
Line 18:		Line 18:


	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 [[~~FICS~~\| FCIS page]].		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]].

	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:		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:

← Older revision		Revision as of 20:54, 7 January 2010
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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.]		*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.]

	*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.]		*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.]

	*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html\|Lance Davidson's flourescent in situ methods page]		*[http://www.faculty.virginia.edu/davidson/fluor_insitu/fluorescent_in_situ.html \| Lance Davidson's flourescent in situ methods page]