TE antigen retrieval and immunostaining - Revision history

Jakob Suckale: categories

Jakob Suckale — Mon, 02 Nov 2009 19:43:31 GMT

categories

←Older revision		Revision as of 19:43, 2 November 2009
Line 8:		Line 8:
	:* 80% ethanol 1min		:* 80% ethanol 1min
	:* distilled water		:* distilled water
-
	Note: Incubation times and repeats vary between protocols.		Note: Incubation times and repeats vary between protocols.

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	=== Principle ===		=== Principle ===
-
	* aldehyde treatment ([[Polymethanal (paraformaldehyde)\|paraformaldehyde]], glutaraldehyde) chemically fixes cells and tissue		* aldehyde treatment ([[Polymethanal (paraformaldehyde)\|paraformaldehyde]], glutaraldehyde) chemically fixes cells and tissue
	* [[Paraffin embedding and sectioning\|paraffin-embedding]] allows very thin tissue sections to be cut		* [[Paraffin embedding and sectioning\|paraffin-embedding]] allows very thin tissue sections to be cut
Line 40:		Line 38:

	=== Comments ===		=== Comments ===
-
	* Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated.		* Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated.
	* Containers immersed in water baths on the other hand may not reach 95ºC but the temperatures is more constant than in microwave treatments. Use a lid to increase the temperature of the buffer.		* Containers immersed in water baths on the other hand may not reach 95ºC but the temperatures is more constant than in microwave treatments. Use a lid to increase the temperature of the buffer.

	== Immunostaining ==		== Immunostaining ==
-
	Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.		Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.

	== See also ==		== See also ==
-	*
	* [http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm Tris EDTA antigen retrieval at IHCWorld]		* [http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm Tris EDTA antigen retrieval at IHCWorld]
	* [http://www.abcam.com/index.html?pageconfig=resource&rid=11488 Antigen retrieval page at abcam]		* [http://www.abcam.com/index.html?pageconfig=resource&rid=11488 Antigen retrieval page at abcam]
	* [http://www.millipore.com/techpublications/tech1/mcproto165 Antigen retrieval page at Millipore]		* [http://www.millipore.com/techpublications/tech1/mcproto165 Antigen retrieval page at Millipore]
	* [http://books.google.com/books?q=tris%20edta%20antigen%20retrieval&hl=en Google Books search for "tris edta antigen retrieval"]		* [http://books.google.com/books?q=tris%20edta%20antigen%20retrieval&hl=en Google Books search for "tris edta antigen retrieval"]
		+
		+	[[Category:Protocol]]
		+	[[Category:Antibody]]
		+	[[Category:Microscopy]]
		+	[[Category:Protein]]

Jakob Suckale: see also

Jakob Suckale — Mon, 02 Nov 2009 19:42:22 GMT

Jakob Suckale: /* Immunostaining */

Jakob Suckale — Mon, 02 Nov 2009 19:34:37 GMT

Immunostaining

←Older revision		Revision as of 19:34, 2 November 2009
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	== Immunostaining ==		== Immunostaining ==
		+
		+	Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.

Jakob Suckale: /* Deparaffinisation */

Jakob Suckale — Mon, 02 Nov 2009 19:33:49 GMT

Deparaffinisation

←Older revision		Revision as of 19:33, 2 November 2009
Line 8:		Line 8:
	:* 80% ethanol 1min		:* 80% ethanol 1min
	:* distilled water		:* distilled water
		+
		+	Note: Incubation times and repeats vary between protocols.

	== Tris EDTA heat-based antigen retrieval ==		== Tris EDTA heat-based antigen retrieval ==

Jakob Suckale at 19:31, 2 November 2009

Jakob Suckale — Mon, 02 Nov 2009 19:31:43 GMT

←Older revision		Revision as of 19:31, 2 November 2009
Line 2:		Line 2:

	== Deparaffinisation ==		== Deparaffinisation ==
		+	* deparaffinise sections in xylene for 10min
		+	* hydration series
		+	:* 2 changes of 100% ethanol for 3 minutes each
		+	:* 95% ethanol 1min
		+	:* 80% ethanol 1min
		+	:* distilled water

	== Tris EDTA heat-based antigen retrieval ==		== Tris EDTA heat-based antigen retrieval ==
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	=== Steps ===		=== Steps ===
-	* deparaffinise sections in xylene for 10min
-	* hydration series
-	~~:* 2 changes of 100% ethanol for 3 minutes each~~
-	~~:* 95% ethanol 1min~~
-	~~:* 80% ethanol 1min~~
-	~~:* distilled water~~
	* pre-heat steamer or water bath with container with Tris-EDTA Buffer until temperature reaches 95°C (do not boil)		* pre-heat steamer or water bath with container with Tris-EDTA Buffer until temperature reaches 95°C (do not boil)
	* immerse slides, cover with lid, incubate for 10-40 minutes (optimal incubation time depends on tissue and antibody)		* immerse slides, cover with lid, incubate for 10-40 minutes (optimal incubation time depends on tissue and antibody)

Jakob Suckale: /* Comments */

Jakob Suckale — Mon, 02 Nov 2009 19:31:04 GMT

Comments

←Older revision		Revision as of 19:31, 2 November 2009
Line 38:		Line 38:

	=== Comments ===		=== Comments ===
		+
		+	* Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated.
		+	* Containers immersed in water baths on the other hand may not reach 95ºC but the temperatures is more constant than in microwave treatments. Use a lid to increase the temperature of the buffer.

	== Immunostaining ==		== Immunostaining ==

Jakob Suckale: /* Tris EDTA heat-based antigen retrieval */

Jakob Suckale — Mon, 02 Nov 2009 19:29:39 GMT

Tris EDTA heat-based antigen retrieval

←Older revision		Revision as of 19:29, 2 November 2009
Line 13:		Line 13:

	=== Material ===		=== Material ===
-
	'''Tris-EDTA Buffer'''		'''Tris-EDTA Buffer'''
	final concentration: 10mM Tris base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0:		final concentration: 10mM Tris base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0:
-
	* 1.21g [[Tris]] final 10mM		* 1.21g [[Tris]] final 10mM
	* 0.37g [[EDTA]] final 1mM		* 0.37g [[EDTA]] final 1mM
Line 23:		Line 21:
	* 0.5ml [[Tween 20]] final 0.05% (for 1x)		* 0.5ml [[Tween 20]] final 0.05% (for 1x)

-	The pH is typically around 9.0. Store the solution at room temperature for ~~3 months~~ or at 4ºC for ~~longer~~.	+	The pH is typically around 9.0 with adjustment. Store the solution at room temperature for several weeks or at 4ºC for months.

	This buffer is basically a [[TE]] buffer with added Tween 20.		This buffer is basically a [[TE]] buffer with added Tween 20.

	=== Steps ===		=== Steps ===
-
	* deparaffinise sections in xylene for 10min		* deparaffinise sections in xylene for 10min
	* hydration series		* hydration series
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	* remove the container and allow to cool at RT for 20 minutes		* remove the container and allow to cool at RT for 20 minutes
	* rinse sections in PBS Tween 20 for 2x2 min		* rinse sections in PBS Tween 20 for 2x2 min
-
-	Note: Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated. Containers immersed in water baths on the other hand may not reach 95ºC. The lid can make a big difference.

	=== Comments ===		=== Comments ===

	== Immunostaining ==		== Immunostaining ==

Jakob Suckale: /* Steps */

Jakob Suckale — Mon, 02 Nov 2009 19:28:29 GMT

Steps

←Older revision		Revision as of 19:28, 2 November 2009
Line 28:		Line 28:

	=== Steps ===		=== Steps ===
		+
		+	* deparaffinise sections in xylene for 10min
		+	* hydration series
		+	:* 2 changes of 100% ethanol for 3 minutes each
		+	:* 95% ethanol 1min
		+	:* 80% ethanol 1min
		+	:* distilled water
		+	* pre-heat steamer or water bath with container with Tris-EDTA Buffer until temperature reaches 95°C (do not boil)
		+	* immerse slides, cover with lid, incubate for 10-40 minutes (optimal incubation time depends on tissue and antibody)
		+	* remove the container and allow to cool at RT for 20 minutes
		+	* rinse sections in PBS Tween 20 for 2x2 min
		+
		+	Note: Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated. Containers immersed in water baths on the other hand may not reach 95ºC. The lid can make a big difference.

	=== Comments ===		=== Comments ===

	== Immunostaining ==		== Immunostaining ==

Jakob Suckale: /* Material */

Jakob Suckale — Mon, 02 Nov 2009 19:05:30 GMT

Material

←Older revision		Revision as of 19:05, 2 November 2009
Line 13:		Line 13:

	=== Material ===		=== Material ===
		+
		+	'''Tris-EDTA Buffer'''
		+	final concentration: 10mM Tris base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0:
		+
		+	* 1.21g [[Tris]] final 10mM
		+	* 0.37g [[EDTA]] final 1mM
		+	* distilled water to 1000 ml
		+	(or 100 ml to make 10x)
		+	* 0.5ml [[Tween 20]] final 0.05% (for 1x)
		+
		+	The pH is typically around 9.0. Store the solution at room temperature for 3 months or at 4ºC for longer.
		+
		+	This buffer is basically a [[TE]] buffer with added Tween 20.

	=== Steps ===		=== Steps ===

Jakob Suckale: /* Principle */

Jakob Suckale — Mon, 02 Nov 2009 18:49:10 GMT

Principle

←Older revision		Revision as of 18:49, 2 November 2009
Line 6:		Line 6:

	=== Principle ===		=== Principle ===
		+
		+	* aldehyde treatment ([[Polymethanal (paraformaldehyde)\|paraformaldehyde]], glutaraldehyde) chemically fixes cells and tissue
		+	* [[Paraffin embedding and sectioning\|paraffin-embedding]] allows very thin tissue sections to be cut
		+	* however, some antibodies don't work well after aldehyde fixation and paraffin embedding as opposed to other fixatives and cryosections
		+	* [[antigen retrieval]] methods can improve antibody binding by reversing chemical modification of epitopes or by unfolding/refolding epitopes

	=== Material ===		=== Material ===

←Older revision		Revision as of 19:42, 2 November 2009
Line 47:		Line 47:

	Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.		Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.
		+
		+	== See also ==
		+	*
		+	* [http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm Tris EDTA antigen retrieval at IHCWorld]
		+	* [http://www.abcam.com/index.html?pageconfig=resource&rid=11488 Antigen retrieval page at abcam]
		+	* [http://www.millipore.com/techpublications/tech1/mcproto165 Antigen retrieval page at Millipore]
		+	* [http://books.google.com/books?q=tris%20edta%20antigen%20retrieval&hl=en Google Books search for "tris edta antigen retrieval"]