FCIS - Revision history

205.142.197.33 at 18:15, 12 June 2012

2012-06-12T18:15:37Z

← Older revision		Revision as of 18:15, 12 June 2012
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	[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.]

2009-12-22T18:57:46Z

Fluorescence colorimetric in situs

← Older revision		Revision as of 18:57, 22 December 2009
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	Hybridize two probes labeled with different haptens simultaneously (e.g. XAA1-DIG and NaK ATPase-FITC) via the standard protocol. DNP labeled RNA probes are also good.		Hybridize two probes labeled with different haptens simultaneously (e.g. XAA1-DIG and NaK ATPase-FITC) via the standard protocol. DNP labeled RNA probes are also good.

	In the antibody step incubate both AP coupled anti-DIG (Roche) plus HRP coupled anti-FITC (Roche) together (if you used DNP in your RNA, use anti-DNP-HRP, Perkin-Elmer). Wash out antibodies and develop standard BM purple color for as long as you want. Rinse, then develop the tyramide counterstain as described from point 5 onwards. Following a nice long wash (2 days) photograph separately using both reflected light and fluorescence.		In the antibody step incubate both AP coupled anti-DIG (Roche) plus HRP coupled anti-FITC (Roche) together (if you used DNP in your RNA, use anti-DNP-HRP, Perkin-Elmer). Wash out antibodies and develop standard BM purple color for as long as you want. Rinse, then develop the [[FISH_/_Double_FISH\| tyramide counterstain as described]] from point 5 onwards. Following a nice long wash (2 days) photograph separately using both reflected light and fluorescence.

	The overlay is done in Adobe PhotoShop as follows (Victor Gerth and Peter Vize)		The overlay is done in Adobe PhotoShop as follows (Victor Gerth and Peter Vize)

2009-12-22T18:56:40Z

← Older revision		Revision as of 18:56, 22 December 2009
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	In the antibody step incubate both AP coupled anti-DIG (Roche) plus HRP coupled anti-FITC (Roche) together (if you used DNP in your RNA, use anti-DNP-HRP, Perkin-Elmer). Wash out antibodies and develop standard BM purple color for as long as you want. Rinse, then develop the tyramide counterstain as described from point 5 onwards. Following a nice long wash (2 days) photograph separately using both reflected light and fluorescence.		In the antibody step incubate both AP coupled anti-DIG (Roche) plus HRP coupled anti-FITC (Roche) together (if you used DNP in your RNA, use anti-DNP-HRP, Perkin-Elmer). Wash out antibodies and develop standard BM purple color for as long as you want. Rinse, then develop the tyramide counterstain as described from point 5 onwards. Following a nice long wash (2 days) photograph separately using both reflected light and fluorescence.


	The overlay is done in Adobe PhotoShop as follows (Victor Gerth and Peter Vize)		The overlay is done in Adobe PhotoShop as follows (Victor Gerth and Peter Vize)


	# Copy green flourescent image to new image.		# Copy green flourescent image to new image.
	# Using the new image paste the green channel into the red channel.		# Using the new image paste the green channel into the red channel.

2009-12-22T18:55:19Z

@@ Line 1: / Line 1: @@
-[[File:FCIS-p big.jpg|200px|thumb|left|alt text]]
+===Fluorescence colorimetric in situs===
-[[File:FCIS-f_big.jpg|200px|thumb|left|alt text]]
+Based on Zhou, X. and Vize, P.D. (2004). [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15223337| Developmental Biology 271: 322-338.]
-[[File:FCIS_big.jpg|200px|thumb|left|alt text]]
+A combination of standard BM purple staining along with a FITC-tyramide or Cy3-tyramide counterstain. The purple precipitate will mask the fluorescence, and the masking shows where your expression is located. For example, compare the images of kidneys stained with XAA1, which is only active in a tiny part of the pronephros. On its own this would be difficult to interpret. The middle panel shows a general pronephric FISH counterstain (sodium potassium ATPase), and the right panel an overlay. Click on the images to enlarge. From the FCIS image the localized pronephric expression is very obvious.

2009-12-22T18:51:17Z

← Older revision		Revision as of 18:51, 22 December 2009
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	[[File:FCIS-p big.jpg\|200px\|thumb\|left\|alt text]]		[[File:FCIS-p big.jpg\|200px\|thumb\|left\|alt text]]
	[[FCIS-f_big.jpg\|200px\|thumb\|left\|alt text]]
	[[FCIS_big.jpg\|200px\|thumb\|left\|alt text]]		[[File:FCIS-f_big.jpg\|200px\|thumb\|left\|alt text]]

			[[File:FCIS_big.jpg\|200px\|thumb\|left\|alt text]]

2009-12-22T18:50:09Z

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