X-gal Staining Protocol: Difference between revisions
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==Procedure== | ==Procedure== | ||
(Bruce Conklin and David Sanan, Gladstone/UCSF) | '''(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) | |||
3) Final X-gal Solution: | '''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. | 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:''' | |||
*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== | ==References== | ||
'''Relevant papers and books''' | '''Relevant papers and books''' | ||
No further references available at this time | |||
==Contact== | ==Contact== | ||
Latest revision as of 14:31, 24 June 2009
Overview
List of reagents is not yet complete
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
No further references available at this time
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.
