In situ hybridization and Small Molecules for Xenopus Research: Difference between pages

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'''In this section:'''
==Important small molecule references==
:Nucleotide Stocks
*[http://www.xenbase.org/literature/article.do?method=display&articleId=35021 Database of Small Molecules by Feldman and Hogue 2006]
:5x SP6 Buffer
*[http://www.xenbase.org/literature/article.do?method=display&articleId=47903 Wnt/Notch by Myers and Krieg 2014]
:MEMFA
*[http://www.xenbase.org/literature/article.do?method=display&articleId=970 Ubiquitin-proteasome by Salic and King 2005]
:PTw
:Triethanolamine 0.1 M
:PTw Containing 4% Paraformaldehyde
:Hybridization Buffer
:SSC, 20X
:Denhardt’s solution, 100X
:RNase A
:RNase T1
:4% BMB Blocking Reagent in MAB
:Alkaline Phosphatase Buffer
:Levamisol 1 M
:NBT/BCIP
:PVA Alkaline Phosphatase Buffer
:Bouin’s Fixative
:Bleaching solution (Mayor et al. 1995)


==Alphabetical List of Small Molecules With Literature References==


'''Nucleotide Stocks'''
*[[11-cis retinal]]
*[[18alpha-glycyrretinic acid]]
*[[3,3,5-triiodothyronine (T3)]]
*[[5HT (seratonin)]]
*[[activin]]
*[[AG1296]]
*[[AG1879]]
*[[AGN193109]]
*[[alpha-amanitin]]
*[[anandamide]]
*[[apelin]]
*[[aphidicolin ]]
*[[AZD1480]]
*[[BAPTA-AM]]
*[[beta-xyloside]]
*[[bumetanide]]
*[[calcium ionophore]]
*[[CGP54626 ]]
*[[Chelerythrine]]
*[[chymostatin ]]
*[[concanamycin]]
*[[cycloheximide]]
*[[cyclopamine]]
*[[Cytochalasin B]]
*[[dexamethosone]]
*[[DHT(dihydrotestosterone)]]
*[[Ex-527]]
*[[forskolin]]
*[[fusicoccin]]
*[[gentamycin]]
*[[GM6001]]
*[[heptanol]]
*[[IBMX]]
*[[KT 5720]]
*[[lansoprazol]]
*[[LiCl]]
*[[Mg132]]
*[[mHip]]
*[[MMP-2 inhibitor II]]
*[[nicorandil]]
*[[nifedipine]]
*[[nocodazole]]
*[[NSC-87877]]
*[[o-phenanthroline]]
*[[olomoucine]]
*[[ouabain]]
*[[p-MPPI]]
*[[phaclofen]]
*[[PMA]]
*[[PP2]]
*[[primaquin]]
*[[propythiouracil]]
*[[retinoic acid]]
*[[RS67333]]
*[[RU486]]
*[[SB 203580]]
*[[sodium nitroprusside]]
*[[SP600125 ]]
*[[SU5402]]
*[[SU66056]]
*[[Suramin]]
*[[thyroxine (T4)]]
*[[TTNPB]]
*[[tunicamycin]]
*[[Y27632]]
*[[ZM 447439]]


2.5 mM nucleotide mix with digoxigenin-11 UTP (Boehringer Mannheim 1209 256; 25 moles)
==List of Molecules Affecting Specific Pathways==
===cAMP===
[[IBMX]]
===ERK/MAPK===
===JAK/STAT===
*[[AZD1480]]
===JNK===
[[SP600125]]


:10 mM CTP - 10 ul
===PDGF===
:10 mM GTP - 10 ul
*[[AG1296]]
:10 mM ATP - 10 ul
===Retinoic Acid===
:10 mM UTP - 6.5 ul
*[[AGN193109]]
:10 mM dig-11UTP - 3.5 ul
*[[retinoic acid]]
*[[TTNPB]]
===Shh===
*[[cyclopamine]]
*[[mHip]]
===TGF-beta/BMP===
===Wnt===
*[[LiCl]]


Alternatively, prepare the whole tube of dig-UTP (25 moles; Boehringer Mannheim 1209 256) at one time to made a 2.5 mM stock.
===Cytoskeletal Inhibitors===
*[[Cytochalasin B]]
===Epigenetics===
[[Ex-527]]
===Protease Inhibitors===
*[[chymostatin]]
*[[GM6001|GM6001 (MMP inhibitor)]]
*[[o-phenanthroline|o-phenanthroline(MMP inhibitor)]]


:100 mM CTP - 7.1 ul
:100 mM GTP - 7.1 ul
:7100 mM ATP  - 1 ul
:100 mM UTP 4.6 ul
:10 mM dig-UTP 25
:DEPC-treated water 234 ul


===Small Molecules Studied in Oocytes Found Via Textpresso===
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 M119]
*[http://www.xenbase.org/literature/article.do?method=display&articleId=42068 GBG or GBetaGamma or G Protein Beta Gamma]
*[[LY-294002| LY-294002 inhibits PI3K and autophagy]]


'''dNTPs 10 mM'''
===Textpresso Small Molecules to Research===
 
*[http://www.xenbase.org/literature/article.do?method=display&articleId=43038 importazole inhibits importin]
dNTPs (Roche), 100 mM (250 ul each)
 
Mix 250 µl each dNTP = 1000µl
Add 1500µl ddH20 for final 2500µl of 10 mM solution.
 
Make 25-50 ul aliquots in eppendorf tubes. Store at -20C.
 
 
'''5x SP6 Buffer'''
:200 mM Tris (pH 7.5)
:30 mM MgCl2
:20 mM spermidine
 
Autoclave and store frozen in aliquots.
 
 
'''MEMFA''' (MOPS/EGTA/Magnesium Sulfate/Formaldehyde Buffer)
 
 
{|border="1" cellpadding="5" cellspacing="0" align="center"
|-
|
! scope="col" style="background:#efefef;" | 1X
! scope="col" style="background:#efefef;" | 10X
! scope="col" style="background:#efefef;" | for 1 L 10X
|-
! scope="row" style="background:#efefef;" | MOPS (pH 7.4)
|0.1 M
|1.0 M
|209.27 g
|-
! scope="row" style="background:#efefef;" | EGTA
|2 mM
|20 mM
|7.608 g
|-
! scope="row" style="background:#efefef;" | MgSO4
|1 mM
|10 mM
|2.46 g
|-
! scope="row" style="background:#efefef;" | Formaldehyde
|3.7%
|
|
|}
 
* Prepare a 10 X solution without the formaldehyde for storage. Add fresh formaldehyde(1/10 volume of standard 30% stock) prior to use. The solution will turn yellow with age and when autoclaved.
 
 
'''PTw'''
 
1X PBS(phosphate buffered saline)
 
0.1% Tween-20
 
 
'''Triethanolamine 0.1 M ''' (pH7.0-8.0; Sigma T 1502)
 
For 1 L:
Dissolve 18.6 g triethanolamine in DEPC water, adjust pH with 8 pellets NaOH. Check pH with drops on pH paper. Adjust volume, store at RT. Do not expose the solution to a pH meter of dubious cleanliness.
 
 
'''PTw Containing 4% Paraformaldehyde'''
 
:1 volume of 20% paraformaldehyde
:4 volumes of PTw
 
Note: To made 20% paraformaldehyde in water, neutralize the cloudy solution with NaOH and then heat at 65 deg C, with shaking, until clear, Store at -20 dec C.
 
 
'''Hybridization Buffer'''
 
:50% formamide, redistilled (BRL 5515UB)
:5x SSC
:1 mg/ml Torula RNA (Type IX, Sigma R 3629)
:100 ug/ml heparin (Sigma H 3125)
:1x Denfardt's solution
:0.1% Tween 20 (Sigma P 1379)
:0.1% CHAPS )Sigma C 3023)
:10 mM EDTA
 
Note: Prepare Torula RNA in DPC-treated water. Store in 1 ml aliquots at -20 deg C. It is normal for this solution to be brown and thus it needs no further processing.
 
For 4 L bottle:
:2 L Formamide
:1 L 20x SSC
:4 g Torula RNA
:0.4 g Heparin
:40 ml Denhardt’s solution
:4 ml Tween 20
:4 g CHAPS
Dissolve in dH2O, adjust volume. Store at -20C.
 
 
'''SSC, 20X''' stock solution
 
:175.3 g NaCl
:88.2 g sodium citrate
 
Dissolve in 800 ml of water, adjust pH to 7.0 with NaOH, and adjust volume to 1 liter.
 
For 4 L bottle:
:NaCl 701.2 g
:Sodium Citrate 352.8 g
 
Dissolve in 3.3 L dH2O, adjust pH 7.0, and volume. Store at RT.
 
 
'''Denhardt’s solution, 100X'''
 
:2% BSA (ICN 810661)
:2% polyvinylpyrrolidone (PVP-40 Sigma)
:2% Ficoll 400 (Pharmacia)
 
For 100 ml 100X:
:BSA 2 g
:PVP-40 2 g
:Ficoll 2 g
 
Add a small amount of water to ingredients, make a slurry, then dilute. Store at -20C.
 
 
'''RNase A''' (Sigma R 5000)
 
Dissolve 10mg/ml in TE (pH 7.8) and boil for 10 min before use. Store aliquots at -20C.
 
 
'''RNase T1'''(Sigma R8251)
 
Dissolve 10,000 units/ ml (1000X) in 0.1 M sodium acetate at pH 5.5.
 
Boil for 10 min before use.
 
Store aliquots at -20C. While in use keep at 4C, avoid repeated thawing and freezing.
 
 
'''Maleic Acid Buffer (MAB), 10X'''
 
:100 mM maleic acid (Sigma M 0357)
: 150 mM NaCl (pH 7.5)
 
This solution is used for numerous washes so make up liter quantities.
 
For 4 L bottle:
:Maleic acid 464.4 g
:350.64 NaCl
 
Start dissolving by adding around 280-320 g or more NaOH pellets. Add more pellets during the day as they dissolve. Procedure takes all day. Adjust pH to 7.5. Adjust volume. Store at RT.
 
 
'''4% BMB Blocking Reagent in MAB'''
 
Dissolve 10g BMB in 100 ml 1X MAB or 50g BMB (the whole bottle) in 500 ml 1X MAB(see above for MAB).
 
Make 50 ml aliquots in Falcon tubes. Store at -20C.
 
 
'''Alkaline Phosphatase Buffer'''
 
:100 mM Tris (pH 9.5)
:50 mM MgCl2
:100 mM NaCl
:0.1% Tween 20 (Sigma P 1379)
2 mM levamisol (Sigma)
 
For 1 L:
:100 ml 1 M Tris pH 9.5
:50 ml 1 M MgCl2
:25 ml 4 M NaCl
:1 ml Tween-20
:2 ml 1 M Levamisol
 
Add fresh levamisol just before use. This reagent inhibits endogenous phosphatase. Alkaline phosphatase buffer can be stored, but magnesium hydroxide tens to precipitate over time. A slight misty precipitate has no effect on the reaction.
 
 
'''Levamisol 1 M'''
 
Dissolve Levamisol (204.292 g/L) in dH2O, adjust volume, store at -20C.
 
 
'''NBT/BCIP'''
 
Nitro blue tetrazolium (NBT): 75 mg/ml in 70% dimethylformamide
 
5-bromo-4-chloro--3-indolyl-phosphate (BCIP): 50 mg/ml in 100% dimethyl-formamide
 
 
'''PVA Alkaline Phosphatase Buffer'''
 
Alkaline posphatase buffer (see above) containing 10% polyvinyl alcohol (Aldrich 36,313-8)
 
Dissolve PVA (98-99% hydrolyzed, average m.w. 31,000-50,000) to a final concentration of 10% alkaline phosphatase buffer. Heat the solution to help dissolve the PVA. PVA tends to precipitate during storage, so do not store this before for more than a few days. The final buffer is quite viscous, and this the NBT, BCIP, and levamisole must be mixed thoroughly before adding it to embryos. The embryos must also be washed well after staining, to prevent precipitation of PVA in methanol.
 
 
 
'''Bouin’s Fixative'''
 
To make up to 100 ml of Bouin's fix, dissolve 1 g of picric acid in 70 ml of water and then add 25 ml of 37% formaldehyde and 5 ml of glacial acetic acid.
 
For 50 ml:
35 ml Piric acid
12.5 ml Formaldehyde
2.5 ml glacial acetic acid
Adjust volume, store at RT.
 
 
'''Bleaching solution (Mayor et al. 1995)'''
 
1% H202
5% formamide
0.5 X SSC(standard saline citrate)

Revision as of 11:08, 2 June 2015

Important small molecule references

Alphabetical List of Small Molecules With Literature References

List of Molecules Affecting Specific Pathways

cAMP

IBMX

ERK/MAPK

JAK/STAT

JNK

SP600125

PDGF

Retinoic Acid

Shh

TGF-beta/BMP

Wnt

Cytoskeletal Inhibitors

Epigenetics

Ex-527

Protease Inhibitors


Small Molecules Studied in Oocytes Found Via Textpresso

Textpresso Small Molecules to Research