X-gal Staining Protocol

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(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...)
Current revision (17:31, 24 June 2009) (view source)
(References)
 
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==Overview==
==Overview==
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Replace this sentence with a brief description of the protocol and its goal.
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List of reagents is not yet complete
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==Materials==
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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.
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*supply 1 (i.e. tubes of a certain size? spreaders?)
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*reagent 1
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*X μL reagent 2
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**component A (reagent 2 is made up of multiple components)
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**component B
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*equipment 1
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*equipment 2
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==Procedure==
==Procedure==
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(Bruce Conklin and David Sanan, Gladstone/UCSF)
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'''(Bruce Conklin and David Sanan, Gladstone/UCSF)
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Materials:
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    * Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
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Materials:'''
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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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    * 1X PBS
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    * X-gal (Boehringer Mannheim #745-740)
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    * DMSO
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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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    * Pap pen (Electron Microscopy Sciences #22303)
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    * Nuclear Fast Red ( also called Kernechtrot)
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    * Gel Mount (Biomeda, M01)
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      Solutions:
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      1) Solution A:
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* Potassium Ferrocyanide Crystalline (Fisher Scientific #P232-500
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      5mM Potassium Ferricyanide Crystalline
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* Potassium Ferrocyanide Trihydrate (Fisher Scientific #P236-500)
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      5mM Potassium Ferricyanide Trihydrate
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* Magnesium Chloride (Fisher Scientific #M33-500)
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      2mM Magnesium Chloride
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* 1X PBS
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      in 1 X PBS
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* X-gal (Boehringer Mannheim #745-740)
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      (stored at 4 deg, protected from light, then warm to 37 degrees C prior to using)
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* DMSO
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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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* Pap pen (Electron Microscopy Sciences #22303)
 +
* Nuclear Fast Red ( also called Kernechtrot)
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* Gel Mount (Biomeda, M01)
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'''Solutions:'''
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2) X-gal Stock Solution (40X):
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'''1) Solution A:'''
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40 mg/ml in DMSO (100 mg in 2.5 ml DMSO)
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*5mM Potassium Ferricyanide Crystalline
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(store at -20 deg, protected from light)
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*5mM Potassium Ferricyanide Trihydrate
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*2mM Magnesium Chloride 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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3) Final X-gal Solution:
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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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*(store at -20 deg, protected from light)
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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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*2. Let section dry completely onto slide
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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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*5. Rinse with 1X PBS
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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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*8. Wash as in step 6, then cover slip with Gel Mount
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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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  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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  5. Rinse with 1X PBS
 
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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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  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.  See the [[OpenWetWare:Biblio]] page for more information. -->
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<biblio>
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No further references available at this time
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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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