X-gal Staining Protocol

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(Procedure)
Current revision (17:31, 24 June 2009) (view source)
(References)
 
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Materials:'''
Materials:'''
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    * Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
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* Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
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    * Potassium Ferrocyanide Trihydrate (Fisher Scientific #P236-500)
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* Potassium Ferrocyanide Trihydrate (Fisher Scientific #P236-500)
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    * Magnesium Chloride (Fisher Scientific #M33-500)
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* Magnesium Chloride (Fisher Scientific #M33-500)
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    * 1X PBS
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* 1X PBS
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    * X-gal (Boehringer Mannheim #745-740)
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* X-gal (Boehringer Mannheim #745-740)
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    * DMSO
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* DMSO
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    * 2% Formaldehyde 0.2% Gluteraldehyde in 1 X PBS
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* 2% Formaldehyde 0.2% Gluteraldehyde in 1 X PBS
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    * Superfrost/ Plus Microscope Slides (Fisher #12-550-15)
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* Superfrost/ Plus Microscope Slides (Fisher #12-550-15)
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    * Pap pen (Electron Microscopy Sciences #22303)
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* Pap pen (Electron Microscopy Sciences #22303)
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    * Nuclear Fast Red ( also called Kernechtrot)
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* Nuclear Fast Red ( also called Kernechtrot)
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    * Gel Mount (Biomeda, M01)
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* Gel Mount (Biomeda, M01)
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       Solutions:
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'''Solutions:'''
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      1) Solution A:
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'''1) Solution A:'''
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      5mM Potassium Ferricyanide Crystalline
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*5mM Potassium Ferricyanide Crystalline
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      5mM Potassium Ferricyanide Trihydrate
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*5mM Potassium Ferricyanide Trihydrate
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      2mM Magnesium Chloride
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*2mM Magnesium Chloride in 1 X PBS
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      in 1 X PBS
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*(stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)
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      (stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)
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2) X-gal Stock Solution (40X):
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'''2) X-gal Stock Solution (40X):'''
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40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)
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*40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)
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(store at -20 deg, protected from light)
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*(store at -20 deg, protected from light)
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3) Final X-gal Solution:
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'''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)
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4) Formalin/Glutaraldehyde fixative:
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'''4) Formalin/Glutaraldehyde fixative:'''
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12.3 ml distilled water
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*12.3 ml distilled water
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10.0 ml 1% glutaraldehyde
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*10.0 ml 1% glutaraldehyde
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2.7 ml formaldehyde stock (37%)
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*2.7 ml formaldehyde stock (37%)
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25.0 ml x2 saline buffer
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*25.0 ml x2 saline buffer
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Staining Protocol:
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'''Staining Protocol:'''
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  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.
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*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.
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  2. Let section dry completely onto slide
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*2. Let section dry completely onto slide
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  3. Rinse with 1X PBS
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*3. Rinse with 1X PBS
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  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)
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*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)
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  5. Rinse with 1X PBS
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*5. Rinse with 1X PBS
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  6. Wash 2 X 2'' with deionized H2O.
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*6. Wash 2 X 2'' with deionized H2O.
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  7. Counterstain 3'' with Nuclear Fast Red
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*7. Counterstain 3'' with Nuclear Fast Red
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  8. Wash as in step 6, then cover slip with Gel Mount
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*8. Wash as in step 6, then cover slip with Gel Mount
==References==
==References==
'''Relevant papers and books'''
'''Relevant papers and books'''
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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.
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No further references available at this time
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<biblio>
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#Goldbeter-PNAS-1981 pmid=6947258
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#Jacob-JMB-1961 pmid=13718526
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#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>
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==Contact==
==Contact==

Current revision

Contents

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.


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