X-gal Staining Protocol - Revision history

Elana Urbansky: /* References */

2009-06-24T21:31:36Z

References

←Older revision		Revision as of 21:31, 24 June 2009
Line 56:		Line 56:
	'''Relevant papers and books'''		'''Relevant papers and books'''

-	~~If this protocol has papers or books associated with it, list those~~ references ~~here. Below is an example for formatting purposes. See the [[OpenWetWare:Biblio]] page for more information.~~	+	No further references available at this time
-	~~<biblio>~~	+
-	~~#Goldbeter-PNAS-1981 pmid=6947258~~	+
-	~~#Jacob-JMB-1961 pmid=13718526~~	+
-	~~#Ptashne-Genetic-Switch isbn=0879697164~~	+
-	~~</biblio>~~	+

	==Contact==		==Contact==

Elana Urbansky: /* Procedure */

2009-06-24T21:31:13Z

Procedure

←Older revision		Revision as of 21:31, 24 June 2009
Line 21:		Line 21:

	'''Solutions:'''		'''Solutions:'''
-	'''	+
-	*1) Solution A:'''	+	'''1) Solution A:'''
	*5mM Potassium Ferricyanide Crystalline		*5mM Potassium Ferricyanide Crystalline
	*5mM Potassium Ferricyanide Trihydrate		*5mM Potassium Ferricyanide Trihydrate
Line 28:		Line 28:
	*(stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)		*(stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)

-	'''*2) X-gal Stock Solution (40X):'''	+	'''2) X-gal Stock Solution (40X):'''
	*40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)		*40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)
	*(store at -20 deg, protected from light)		*(store at -20 deg, protected from light)

-	'''*3) Final X-gal Solution:'''	+	'''3) Final X-gal Solution:'''
	Dilute X-gal stock solution 1:40 in Solution A.		Dilute X-gal stock solution 1:40 in Solution A.
	(first warm Solution A to 37 degrees C to prevent precipitation of X-gal)		(first warm Solution A to 37 degrees C to prevent precipitation of X-gal)

-	'''*4) Formalin/Glutaraldehyde fixative:'''	+	'''4) Formalin/Glutaraldehyde fixative:'''
	*12.3 ml distilled water		*12.3 ml distilled water
	*10.0 ml 1% glutaraldehyde		*10.0 ml 1% glutaraldehyde

Elana Urbansky: /* Procedure */

2009-06-24T21:30:19Z

Procedure

←Older revision		Revision as of 21:30, 24 June 2009
Line 8:		Line 8:
	Materials:'''		Materials:'''

-	* Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500	+	* Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
-	* Potassium Ferrocyanide Trihydrate (Fisher Scientific #P236-500)	+	* Potassium Ferrocyanide Trihydrate (Fisher Scientific #P236-500)
-	* Magnesium Chloride (Fisher Scientific #M33-500)	+	* Magnesium Chloride (Fisher Scientific #M33-500)
-	* 1X PBS	+	* 1X PBS
-	* X-gal (Boehringer Mannheim #745-740)	+	* X-gal (Boehringer Mannheim #745-740)
-	* DMSO	+	* DMSO
-	* 2% Formaldehyde 0.2% Gluteraldehyde in 1 X PBS	+	* 2% Formaldehyde 0.2% Gluteraldehyde in 1 X PBS
-	* Superfrost/ Plus Microscope Slides (Fisher #12-550-15)	+	* Superfrost/ Plus Microscope Slides (Fisher #12-550-15)
-	* Pap pen (Electron Microscopy Sciences #22303)	+	* Pap pen (Electron Microscopy Sciences #22303)
-	* Nuclear Fast Red ( also called Kernechtrot)	+	* Nuclear Fast Red ( also called Kernechtrot)
-	* Gel Mount (Biomeda, M01)	+	* Gel Mount (Biomeda, M01)
-	Solutions:	+
		+	'''Solutions:'''
		+	'''
		+	*1) Solution A:'''
		+	*5mM Potassium Ferricyanide Crystalline
		+	*5mM Potassium Ferricyanide Trihydrate
		+	*2mM Magnesium Chloride in 1 X PBS
		+	*(stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)

-	1) Solution A:	+	'''*2) X-gal Stock Solution (40X):'''
-	~~5mM Potassium Ferricyanide Crystalline~~	+	*40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)
-	~~5mM Potassium Ferricyanide Trihydrate~~	+	*(store at -20 deg, protected from light)
-	~~2mM Magnesium Chloride~~	+
-	in ~~1 X PBS~~	+
-	(~~stored~~ at 4 deg, protected from light~~, then warm to 37 degrees C prior to using~~)	+

-	~~2) X-gal Stock Solution (40X):~~	+	'''*3) Final X-gal Solution:'''
-	~~40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)~~	+
-	~~(store at -20 deg, protected from light)~~	+
-		+
-	3) Final X-gal Solution:	+
	Dilute X-gal stock solution 1:40 in Solution A.		Dilute X-gal stock solution 1:40 in Solution A.
	(first warm Solution A to 37 degrees C to prevent precipitation of X-gal)		(first warm Solution A to 37 degrees C to prevent precipitation of X-gal)

-	4) Formalin/Glutaraldehyde fixative:	+	'''*4) Formalin/Glutaraldehyde fixative:'''
-	12.3 ml distilled water	+	*12.3 ml distilled water
-	10.0 ml 1% glutaraldehyde	+	*10.0 ml 1% glutaraldehyde
-	2.7 ml formaldehyde stock (37%)	+	*2.7 ml formaldehyde stock (37%)
-	25.0 ml x2 saline buffer	+	*25.0 ml x2 saline buffer

-	Staining Protocol:	+	'''Staining Protocol:'''

-	1. Cut 10 micrometers cryostat sections onto pap-penned slides from fresh-frozen tissues. Immerse immediately into cold formaldehyde/gluteraldehyde 5 minutes, then rinse in dH20 for 60 seconds.	+	*1. Cut 10 micrometers cryostat sections onto pap-penned slides from fresh-frozen tissues. Immerse immediately into cold formaldehyde/gluteraldehyde 5 minutes, then rinse in dH20 for 60 seconds.
-	2. Let section dry completely onto slide	+	*2. Let section dry completely onto slide
-	3. Rinse with 1X PBS	+	*3. Rinse with 1X PBS
-	4. Apply Final X-gal Solution to sections and incubate at 37 deg; for 30 minutes to 24 hours (Check sections under microscope every 2 hours for development of blue staining)	+	*4. Apply Final X-gal Solution to sections and incubate at 37 deg; for 30 minutes to 24 hours (Check sections under microscope every 2 hours for development of blue staining)
-	5. Rinse with 1X PBS	+	*5. Rinse with 1X PBS
-	6. Wash 2 X 2'' with deionized H2O.	+	*6. Wash 2 X 2'' with deionized H2O.
-	7. Counterstain 3'' with Nuclear Fast Red	+	*7. Counterstain 3'' with Nuclear Fast Red
-	8. Wash as in step 6, then cover slip with Gel Mount	+	*8. Wash as in step 6, then cover slip with Gel Mount

	==References==		==References==

Elana Urbansky: /* Procedure */

2009-06-24T21:24:38Z

Procedure

←Older revision		Revision as of 21:24, 24 June 2009
Line 4:		Line 4:

	==Procedure==		==Procedure==
-	(Bruce Conklin and David Sanan, Gladstone/UCSF)	+	'''(Bruce Conklin and David Sanan, Gladstone/UCSF)
-	Materials:	+
		+	Materials:'''

	* Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500		* Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
Line 50:		Line 51:
	6. Wash 2 X 2'' with deionized H2O.		6. Wash 2 X 2'' with deionized H2O.
	7. Counterstain 3'' with Nuclear Fast Red		7. Counterstain 3'' with Nuclear Fast Red
-	8. Wash as in step 6, then cover slip with Gel Mount	+	8. Wash as in step 6, then cover slip with Gel Mount
		+
	==References==		==References==
	'''Relevant papers and books'''		'''Relevant papers and books'''

Elana Urbansky at 19:39, 16 June 2009

2009-06-16T19:39:28Z

←Older revision		Revision as of 19:39, 16 June 2009
Line 1:		Line 1:
	==Overview==		==Overview==

-		+	List of reagents is not yet complete
-	List of ~~Reagents~~ is not yet complete	+

	==Procedure==		==Procedure==

Elana Urbansky at 19:10, 16 June 2009

2009-06-16T19:10:33Z

←Older revision		Revision as of 19:10, 16 June 2009
Line 1:		Line 1:
	==Overview==		==Overview==

-	~~Replace this sentence with a brief description of the protocol and its goal.~~

-	~~==Materials==~~	+	List of Reagents is not yet complete
-	List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.	+
-		+
-	*supply 1 (i.e. tubes of ~~a certain size? spreaders?)~~	+
-	*reagent 1	+
-	*X μL reagent 2	+
-	**component A (reagent 2 is ~~made up of multiple components)~~	+
-	**component B	+
-	*equipment 1	+
-	*equipment 2	+

	==Procedure==		==Procedure==

BruceConklin at 17:13, 30 July 2008

2008-07-30T17:13:09Z

←Older revision		Revision as of 17:13, 30 July 2008
Line 64:		Line 64:
	==References==		==References==
	'''Relevant papers and books'''		'''Relevant papers and books'''
-	~~<!--~~ If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. ~~-->~~	+
		+	If this protocol has papers or books associated with it, list those references here. Below is an example for formatting purposes. See the [[OpenWetWare:Biblio]] page for more information.
	<biblio>		<biblio>
	#Goldbeter-PNAS-1981 pmid=6947258		#Goldbeter-PNAS-1981 pmid=6947258

BruceConklin at 00:12, 26 July 2008

2008-07-26T00:12:17Z

←Older revision		Revision as of 00:12, 26 July 2008
Line 1:		Line 1:
-
	==Overview==		==Overview==

BruceConklin: New page: ==Overview== Replace this sentence with a brief description of the protocol and its goal. ==Materials== List reagents, supplies and equipment necessary to perform the protocol here. Fo...

2008-07-26T00:10:43Z

New page: ==Overview== Replace this sentence with a brief description of the protocol and its goal. ==Materials== List reagents, supplies and equipment necessary to perform the protocol here. Fo...

New page

==Overview==

Replace this sentence with a brief description of the protocol and its goal.

==Materials==
List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.

*supply 1 (i.e. tubes of a certain size? spreaders?)
*reagent 1
*X μL reagent 2
**component A (reagent 2 is made up of multiple components)
**component B
*equipment 1
*equipment 2

==Procedure==
(Bruce Conklin and David Sanan, Gladstone/UCSF)
Materials:

* Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
* Potassium Ferrocyanide Trihydrate (Fisher Scientific #P236-500)
* Magnesium Chloride (Fisher Scientific #M33-500)
* 1X PBS
* X-gal (Boehringer Mannheim #745-740)
* DMSO
* 2% Formaldehyde 0.2% Gluteraldehyde in 1 X PBS
* Superfrost/ Plus Microscope Slides (Fisher #12-550-15)
* Pap pen (Electron Microscopy Sciences #22303)
* Nuclear Fast Red ( also called Kernechtrot)
* Gel Mount (Biomeda, M01)
Solutions:

1) Solution A:
5mM Potassium Ferricyanide Crystalline
5mM Potassium Ferricyanide Trihydrate
2mM Magnesium Chloride
in 1 X PBS
(stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)

2) X-gal Stock Solution (40X):
40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)
(store at -20 deg, protected from light)

3) Final X-gal Solution:
Dilute X-gal stock solution 1:40 in Solution A.
(first warm Solution A to 37 degrees C to prevent precipitation of X-gal)

4) Formalin/Glutaraldehyde fixative:
12.3 ml distilled water
10.0 ml 1% glutaraldehyde
2.7 ml formaldehyde stock (37%)
25.0 ml x2 saline buffer

Staining Protocol:

1. Cut 10 micrometers cryostat sections onto pap-penned slides from fresh-frozen tissues. Immerse immediately into cold formaldehyde/gluteraldehyde 5 minutes, then rinse in dH20 for 60 seconds.
2. Let section dry completely onto slide
3. Rinse with 1X PBS
4. Apply Final X-gal Solution to sections and incubate at 37 deg; for 30 minutes to 24 hours (Check sections under microscope every 2 hours for development of blue staining)
5. Rinse with 1X PBS
6. Wash 2 X 2'' with deionized H2O.
7. Counterstain 3'' with Nuclear Fast Red
8. Wash as in step 6, then cover slip with Gel Mount
==References==
'''Relevant papers and books'''

<biblio>
#Goldbeter-PNAS-1981 pmid=6947258
#Jacob-JMB-1961 pmid=13718526
#Ptashne-Genetic-Switch isbn=0879697164
</biblio>

==Contact==
*Who has experience with this protocol?

or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].