Paraffin embedding and sectioning - Revision history

Jakob Suckale: /* Steps */

Jakob Suckale — Wed, 30 Sep 2009 14:33:03 GMT

Steps

←Older revision		Revision as of 14:33, 30 September 2009
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	:* embed tissues into paraffin blocks		:* embed tissues into paraffin blocks

-	* trim paraffin blocks to an optimal cutting surface including the sample with a small paraffin frame	+	* '''cut and mount''' sample sections
-	* cut 3-10 µm slices (5 µm is commonly used); use a brush to draw the section onto the knife holder	+	:* trim paraffin blocks to an optimal cutting surface including the sample with a small paraffin frame
-	* place paraffin ribbon or slice in 40-45ºC water bath with a 2nd wet brush (it will expand and wrinkles will vanish)	+	:* cut 3-10 µm slices (5 µm is commonly used); use a brush to draw the section onto the knife holder
-	* fish out swimming paraffin section using glass slides and a brush to position the section	+	:* place paraffin ribbon or slice in 40-45ºC water bath with a 2nd wet brush (it will expand and wrinkles will vanish)
-	* dry sections O/N at 37ºC (lower baking temperatures are better for subsequent antibody detection)	+	:* fish out swimming paraffin section using glass slides and a brush to position the section
		+	:* dry sections O/N at 37ºC (lower baking temperatures are better for subsequent antibody detection)

	* '''rehydrate''' for subsequent methods (paraffin to water):		* '''rehydrate''' for subsequent methods (paraffin to water):

Jakob Suckale at 14:32, 30 September 2009

Jakob Suckale — Wed, 30 Sep 2009 14:32:11 GMT

←Older revision		Revision as of 14:32, 30 September 2009
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	:* rinse in distilled water		:* rinse in distilled water

-	== Tips ==	+	== Tips/Notes ==
	* To cut very thin 1-2µm sections cool the block for 30min at -20ºC and cut using without automatic advancing of the block, relying instead on the temperature expansion of the block. A cutting angle of 15ºC is optimal. More acute angle may not cut at all. Larger angles may break off the section.		* To cut very thin 1-2µm sections cool the block for 30min at -20ºC and cut using without automatic advancing of the block, relying instead on the temperature expansion of the block. A cutting angle of 15ºC is optimal. More acute angle may not cut at all. Larger angles may break off the section.
	* Samples for normal histochemical analysis can be dried faster at 45-60ºC for 6h-O/N. Do not bake sections >25µm at >50ºC or cracks may appear and parts of the sample may fall off during later washes.		* Samples for normal histochemical analysis can be dried faster at 45-60ºC for 6h-O/N. Do not bake sections >25µm at >50ºC or cracks may appear and parts of the sample may fall off during later washes.

Jakob Suckale at 14:30, 30 September 2009

Jakob Suckale — Wed, 30 Sep 2009 14:30:06 GMT

←Older revision		Revision as of 14:30, 30 September 2009
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	* cut fixed tissues into appropriate portions and place in embedding cassettes		* cut fixed tissues into appropriate portions and place in embedding cassettes

-	* dehydrate for paraffin embedding (water to paraffin):	+	* '''dehydrate''' for paraffin embedding (water to paraffin):
	:* 70% ethanol, 2 changes, 1h each		:* 70% ethanol, 2 changes, 1h each
	:* 80% ethanol, 2 changes, 1h each		:* 80% ethanol, 2 changes, 1h each
Line 28:		Line 28:
	* dry sections O/N at 37ºC (lower baking temperatures are better for subsequent antibody detection)		* dry sections O/N at 37ºC (lower baking temperatures are better for subsequent antibody detection)

-	* paraffin to water:	+	* '''rehydrate''' for subsequent methods (paraffin to water):
-	:* ~~deparaffinise sections in~~ 2 changes of xylene, ~~10m~~ each (3 changes for sections >25µm)	+	:* 2 changes of xylene, 3-10 min each (3+ changes for sections >25µm) [deparaffinise]
-	:* ~~re-hydrate in 1-~~2 changes of 100% ethanol, 3 ~~minutes~~ each	+	:* 2 changes of 100% ethanol, 3 min each [re-hydrate]
-	:* ~~1 min~~ 95% ethanol	+	:* 2 changes of 95% ethanol, 3 min each [re-hydrate]
-	~~:* 1~~ min ~~80% ethanol (other protocols employ a single 3min 95% EtOH step instead of 95%+80%)~~	+
	:* rinse in distilled water		:* rinse in distilled water

	== Tips ==		== Tips ==
	* To cut very thin 1-2µm sections cool the block for 30min at -20ºC and cut using without automatic advancing of the block, relying instead on the temperature expansion of the block. A cutting angle of 15ºC is optimal. More acute angle may not cut at all. Larger angles may break off the section.		* To cut very thin 1-2µm sections cool the block for 30min at -20ºC and cut using without automatic advancing of the block, relying instead on the temperature expansion of the block. A cutting angle of 15ºC is optimal. More acute angle may not cut at all. Larger angles may break off the section.
-	* Samples for normal histochemical analysis can be dried faster at 45-60ºC for 6-4h. Do not bake sections >25µm at >50ºC or cracks may appear and parts of the sample may fall off during later washes.	+	* Samples for normal histochemical analysis can be dried faster at 45-60ºC for 6h-O/N. Do not bake sections >25µm at >50ºC or cracks may appear and parts of the sample may fall off during later washes.
		+	* Rehydration series vary a lot between different protocols. Some labs use 95% 1min, 80% 1min or even lower ethanol solutions.
		+	* Paraffin embedding instruments are available. They dehydrate the sample to 100% ethanol and then infiltrate the tissue with xylene and later molten paraffin at 60-70ºC. Avoid paraffinisation temperatures above 65º if you want to label with antibodies later on, since epitopes can be destroyed.

	== See also ==		== See also ==
Line 47:		Line 48:
	=== Books ===		=== Books ===
	* [http://books.google.com/books?id=02mLqzXwStUC&pg=RA1-PA564&dq=paraffin+sections&lr=&as_drrb_is=b&as_minm_is=0&as_miny_is=1995&as_maxm_is=0&as_maxy_is=&as_brr=0&ei=-FPDSvLJDqG8zgSjq_j1Aw#v=onepage&q=paraffin%20sections&f=false Cell biology: a laboratory handbook, Volume 1, by Julio E. Celis]		* [http://books.google.com/books?id=02mLqzXwStUC&pg=RA1-PA564&dq=paraffin+sections&lr=&as_drrb_is=b&as_minm_is=0&as_miny_is=1995&as_maxm_is=0&as_maxy_is=&as_brr=0&ei=-FPDSvLJDqG8zgSjq_j1Aw#v=onepage&q=paraffin%20sections&f=false Cell biology: a laboratory handbook, Volume 1, by Julio E. Celis]
		+	* [http://books.google.com/books?id=vHL7FZHYcLIC&pg=PA163&dq=paraffin+sections&lr=&as_drrb_is=b&as_minm_is=0&as_miny_is=1995&as_maxm_is=0&as_maxy_is=&as_brr=0&ei=ZWbDSuagGJPuygTR0bX7Aw#v=onepage&q=paraffin%20sections&f=false Using antibodies: a laboratory manual, by Edward Harlow and David Lane]

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:Microscopy]]		[[Category:Microscopy]]
	[[Category:In vivo]]		[[Category:In vivo]]

Jakob Suckale: backlink

Jakob Suckale — Wed, 30 Sep 2009 14:13:23 GMT

backlink

←Older revision		Revision as of 14:13, 30 September 2009
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		+	{{back to protocols}}
		+
	Biological samples often need to be solidified to allow fine sectioning. Thin slices improve the access of dyes, probes, and antibodies and reduce the overlay of different cells layers in the z direction. For light microscopy, paraffin wax is the most frequently used hard matrix for cutting.		Biological samples often need to be solidified to allow fine sectioning. Thin slices improve the access of dyes, probes, and antibodies and reduce the overlay of different cells layers in the z direction. For light microscopy, paraffin wax is the most frequently used hard matrix for cutting.

Jakob Suckale: /* Steps */

Jakob Suckale — Wed, 30 Sep 2009 14:12:38 GMT

Steps

←Older revision		Revision as of 14:12, 30 September 2009
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	* paraffin to water:		* paraffin to water:
-	:* deparaffinise sections in 2-3 changes of xylene, 10m each (3 changes ~~of xylene~~ for sections >25µm)	+	:* deparaffinise sections in 2 changes of xylene, 10m each (3 changes for sections >25µm)
-	:* hydrate in 2 changes of 100% ethanol ~~for~~ 3 minutes each	+	:* re-hydrate in 1-2 changes of 100% ethanol, 3 minutes each
-	:* 1x 95% ethanol~~, 1 minute~~	+	:* 1 min 95% ethanol
-	:* 1x 80% ethanol~~, 1 minute~~	+	:* 1 min 80% ethanol (other protocols employ a single 3min 95% EtOH step instead of 95%+80%)
	:* rinse in distilled water		:* rinse in distilled water

Jakob Suckale: tips

Jakob Suckale — Wed, 30 Sep 2009 13:10:59 GMT

tips

←Older revision		Revision as of 13:10, 30 September 2009
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	== Steps ==		== Steps ==
-
	* fix tissues with 4% PFA or other fixatives; fixative volume should be 5-10x of tissue volume		* fix tissues with 4% PFA or other fixatives; fixative volume should be 5-10x of tissue volume
	* cut fixed tissues into appropriate portions and place in embedding cassettes		* cut fixed tissues into appropriate portions and place in embedding cassettes
Line 21:		Line 20:
	:* embed tissues into paraffin blocks		:* embed tissues into paraffin blocks

-	* trim paraffin blocks ~~and~~ cut at 3-10 µm (5 µm is commonly used)	+	* trim paraffin blocks to an optimal cutting surface including the sample with a small paraffin frame
-	* place paraffin ribbon or slice in 40-45ºC water bath	+	* cut 3-10 µm slices (5 µm is commonly used); use a brush to draw the section onto the knife holder
-	* fish out swimming paraffin section using glass slides	+	* place paraffin ribbon or slice in 40-45ºC water bath with a 2nd wet brush (it will expand and wrinkles will vanish)
-	* allow sections to ~~air dry for 30 minutes~~	+	* fish out swimming paraffin section using glass slides and a brush to position the section
-	* ~~bake in 45-50ºC oven overnight~~	+	* dry sections O/N at 37ºC (lower baking temperatures are better for subsequent antibody detection)
-	~~:NEVER go higher than 50ºC for~~ sections ~~thicker than 25µm. Otherwise, sections may crack and fall off during staining~~	+

	* paraffin to water:		* paraffin to water:
Line 35:		Line 33:
	:* rinse in distilled water		:* rinse in distilled water

		+	== Tips ==
		+	* To cut very thin 1-2µm sections cool the block for 30min at -20ºC and cut using without automatic advancing of the block, relying instead on the temperature expansion of the block. A cutting angle of 15ºC is optimal. More acute angle may not cut at all. Larger angles may break off the section.
		+	* Samples for normal histochemical analysis can be dried faster at 45-60ºC for 6-4h. Do not bake sections >25µm at >50ºC or cracks may appear and parts of the sample may fall off during later washes.

	== See also ==		== See also ==
Line 42:		Line 43:
	* [http://en.wikipedia.org/wiki/Paraffin Wikipedia: Paraffin]		* [http://en.wikipedia.org/wiki/Paraffin Wikipedia: Paraffin]

		+	=== Books ===
		+	* [http://books.google.com/books?id=02mLqzXwStUC&pg=RA1-PA564&dq=paraffin+sections&lr=&as_drrb_is=b&as_minm_is=0&as_miny_is=1995&as_maxm_is=0&as_maxy_is=&as_brr=0&ei=-FPDSvLJDqG8zgSjq_j1Aw#v=onepage&q=paraffin%20sections&f=false Cell biology: a laboratory handbook, Volume 1, by Julio E. Celis]

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:Microscopy]]		[[Category:Microscopy]]
	[[Category:In vivo]]		[[Category:In vivo]]

Jakob Suckale: steps

Jakob Suckale — Wed, 30 Sep 2009 12:45:59 GMT

steps

←Older revision		Revision as of 12:45, 30 September 2009
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	== Section thickness ==		== Section thickness ==
	Paraffin sections for light microscopy are typically 5 μm thick. Paraffin wax does not provide a sufficiently hard matrix for cutting thinner slices needed for electron microscopy, typically 80-100 nm thick. Here, epoxy or acrylic resins especially for immunohistochemistry are used.		Paraffin sections for light microscopy are typically 5 μm thick. Paraffin wax does not provide a sufficiently hard matrix for cutting thinner slices needed for electron microscopy, typically 80-100 nm thick. Here, epoxy or acrylic resins especially for immunohistochemistry are used.
		+
		+	== Steps ==
		+
		+	* fix tissues with 4% PFA or other fixatives; fixative volume should be 5-10x of tissue volume
		+	* cut fixed tissues into appropriate portions and place in embedding cassettes
		+
		+	* dehydrate for paraffin embedding (water to paraffin):
		+	:* 70% ethanol, 2 changes, 1h each
		+	:* 80% ethanol, 2 changes, 1h each
		+	:* 95% ethanol, 2 changes, 1h each
		+	:* 100% ethanol, 3 changes, 1h each
		+	:* xylene or substitute (i.e. Clear Rite 3), 3 changes, 1h each
		+	:* paraffin wax (56-58ºC), 2 changes, 1.5h each
		+	:* embed tissues into paraffin blocks
		+
		+	* trim paraffin blocks and cut at 3-10 µm (5 µm is commonly used)
		+	* place paraffin ribbon or slice in 40-45ºC water bath
		+	* fish out swimming paraffin section using glass slides
		+	* allow sections to air dry for 30 minutes
		+	* bake in 45-50ºC oven overnight
		+	:NEVER go higher than 50ºC for sections thicker than 25µm. Otherwise, sections may crack and fall off during staining
		+
		+	* paraffin to water:
		+	:* deparaffinise sections in 2-3 changes of xylene, 10m each (3 changes of xylene for sections >25µm)
		+	:* hydrate in 2 changes of 100% ethanol for 3 minutes each
		+	:* 1x 95% ethanol, 1 minute
		+	:* 1x 80% ethanol, 1 minute
		+	:* rinse in distilled water
		+

	== See also ==		== See also ==

Jakob Suckale: links

Jakob Suckale — Wed, 30 Sep 2009 10:14:21 GMT

links

←Older revision		Revision as of 10:14, 30 September 2009
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	== See also ==		== See also ==
	* [[Griffin:Immunohistochemistry Paraffin\|Griffin lab: Immunohistochemistry on paraffin sections]]		* [[Griffin:Immunohistochemistry Paraffin\|Griffin lab: Immunohistochemistry on paraffin sections]]
-	* [~~http~~:~~//openwetware.org/images/d/d0/Alikprotocols2006.pdf~~ Khademhosseini lab: paraffin embedding]	+	* [[Alik:Paraffin embedding\|Khademhosseini lab: paraffin embedding]]
	* [http://en.wikipedia.org/wiki/Histology#Processing_-_Dehydration.2C_Clearing_and_Infiltration Wikipedia: Histology, section processing]		* [http://en.wikipedia.org/wiki/Histology#Processing_-_Dehydration.2C_Clearing_and_Infiltration Wikipedia: Histology, section processing]
	* [http://en.wikipedia.org/wiki/Paraffin Wikipedia: Paraffin]		* [http://en.wikipedia.org/wiki/Paraffin Wikipedia: Paraffin]

Jakob Suckale: categories

Jakob Suckale — Wed, 30 Sep 2009 09:58:49 GMT

Jakob Suckale: see also

Jakob Suckale — Wed, 30 Sep 2009 09:57:14 GMT

←Older revision		Revision as of 09:58, 30 September 2009
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	* [http://en.wikipedia.org/wiki/Histology#Processing_-_Dehydration.2C_Clearing_and_Infiltration Wikipedia: Histology, section processing]		* [http://en.wikipedia.org/wiki/Histology#Processing_-_Dehydration.2C_Clearing_and_Infiltration Wikipedia: Histology, section processing]
	* [http://en.wikipedia.org/wiki/Paraffin Wikipedia: Paraffin]		* [http://en.wikipedia.org/wiki/Paraffin Wikipedia: Paraffin]
		+
		+
		+	[[Category:Protocol]]
		+	[[Category:Microscopy]]
		+	[[Category:In vivo]]

←Older revision		Revision as of 09:57, 30 September 2009
Line 6:		Line 6:
	== Section thickness ==		== Section thickness ==
	Paraffin sections for light microscopy are typically 5 μm thick. Paraffin wax does not provide a sufficiently hard matrix for cutting thinner slices needed for electron microscopy, typically 80-100 nm thick. Here, epoxy or acrylic resins especially for immunohistochemistry are used.		Paraffin sections for light microscopy are typically 5 μm thick. Paraffin wax does not provide a sufficiently hard matrix for cutting thinner slices needed for electron microscopy, typically 80-100 nm thick. Here, epoxy or acrylic resins especially for immunohistochemistry are used.
		+
		+	== See also ==
		+	* [[Griffin:Immunohistochemistry Paraffin\|Griffin lab: Immunohistochemistry on paraffin sections]]
		+	* [http://openwetware.org/images/d/d0/Alikprotocols2006.pdf Khademhosseini lab: paraffin embedding]
		+	* [http://en.wikipedia.org/wiki/Histology#Processing_-_Dehydration.2C_Clearing_and_Infiltration Wikipedia: Histology, section processing]
		+	* [http://en.wikipedia.org/wiki/Paraffin Wikipedia: Paraffin]