X-gal Staining Protocol

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(Procedure)
(Procedure)
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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
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    * 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==

Revision as of 17:30, 24 June 2009

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

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.

  1. Goldbeter A and Koshland DE Jr. . pmid:6947258. PubMed HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. . pmid:13718526. PubMed HubMed [Jacob-JMB-1961]
  3. Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. isbn:0879697164. [Ptashne-Genetic-Switch]
All Medline abstracts: PubMed HubMed

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