Reagents and Recipes

Fixation

• 50% bleach= 1:1 mix with ddH₂O

• Embryo wash buffer= 8 L ddH₂O, 48 g sodium chloride, 16 ml 20% TritonX-100

• Fixation Recipe options:
  1. Recipe 1=
     • 3 ml fix buffer= 1.3x PBS, 67 mM EGTA pH 8.0
       1 ml 37% formaldehyde solution (methanol stabilized) e.g. Fisher #BP531 (500 ml)
       5 ml heptane
       
     • Final formaldehyde concentration= ~9%.
     • 37% formaldehyde works less well after a few months of the bottle being opened.
  2. Recipe 2=
     • 1 ml fix buffer= 0.5 ml 10x PBS + 0.5 ml 0.5 M EGTA pH 8.0
       4 ml 10% ultrapure formaldehyde (methanol free) e.g. Polysciences #04018 (1 L)
       5 ml heptane
       
     • Final formaldehyde concentration= ~8%.
     • This is a superior fix recipe for this protocol.

Embryo pre-treatment and hybridization

• PBT= 1x PBS, 0.1% Tween-20
  • Use regular autoclaved PBS which is not DEPC-treated.
  • Make a stock of 10% Tween and mix it 1:100 in PBS.
  • Store larger volumes (more than 100 ml) of PBT at 4° C. if it is not used within a few days.

• PBT+ 5% formaldehyde=
  • 1:1 mix of PBT:10% formaldehyde OR
    6:1 mix of PBT:37% formaldehyde
  • Make this solution freshly on the day of the experiment, as it does not keep.

• Proteinase K stock solution= 10 mg/ml in ddH₂O, e.g. Sigma #P-2308 (25 mg)
  • Make very small aliquots (<10 µl) and store frozen at -20° C.
  • Thaw a new aliquot each time to assure consistent activity.
  • Optimize the final concentration for each batch of ProtK: various protocols call for a range of between 4-50 µg/ml final concentration, and a range of digestion times. Determine the optimum working concentration of the stock by performing parallel experiments using different concentrations for a constant digestion time. If the concentration is too low, the signals from the hybridized probes will be reduced, and if it is too high, the embryos will fall apart during hybridization or appear mushy.

• Hybridization solution=
  • 50% formamide (molecular biology grade), e.g. Roche #1 814 320 (500 ml)
    5x SSC (20x SSC stock)
    100 µg/ml fragmented salmon testes DNA (10 mg/ml stock), e.g. Sigma #D-1626
    50 µg/ml heparin (10 mg/ml stock), e.g. Sigma #H-3393
    0.1% Tween
    
  • Molecular biology grade formamide does not need to be de-ionized or filtered before use.
  • Use autoclaved ddH₂O to make up this solution.
  • Some protocols call for 50 µg/ml tRNA as additional carrier and competitor.
  • Adjusting the pH is not necessary unless a higher hybridization temperature (>55° C.) is used. The solution will become more basic at higher temperature, so lowering the pH to between 4.5-6.5 (either with 1 M citric acid or 1 N HCl) will compensate for this change at hybridization temperatures between 60°-70° C.
  • The salmon testes DNA stock solution can be prepared by autoclaving it for 20 minutes after having stirred it into solution.

Reagents for making RNA probes

• This table contains information on the reagents needed to make RNA probes:

  Reagent	Company	Catalog #
  T7 RNA Polymerase(+transcription buffer)	Roche	881 767
  T3 RNA Polymerase(+transcription buffer)	Roche	1 031 163
  Protector RNase Inhibitor	Roche	3 335 399
  Nuclease-free ddH2O	Sigma	W-4502
  10x Digoxigenin(DIG) RNA labeling mix	Roche	1 277 073
  10x Biotin(BIO) RNA labeling mix	Roche	1 685 597
  10x Fluorescein(FITC) RNA labeling mix	Roche	1 685 619

• 10x Dinitrophenyl (DNP) RNA labeling mix= make this solution with:
  • DNP-11-UTP (250 nmol, 10 mM), Perkin Elmer #NEL 555 and
  • Ribonucleoside Triphosphate Set (100 mM NTPs), Roche #1 277 057
  • Use the same NTP concentrations as are in the Roche 10x labeling mixes, including the same relative amounts of the normal and modified UTPs.

• 2x carbonate buffer=
  • 120 mM Na₂CO₃
    80 mM NaHCO₃
    
  • pH to 10.2, aliquot and store at -20° C.

• Stop solution=
  • 0.2 M sodium acetate
  • pH to 6.0 with acetic acid, aliquot and store at -20° C.

Reagents for detecting probes

• This table contains information on the primary probe detection reagents:

  Reagent	Company	Catalog #
  Mouse anti-DIG	Roche	1 333 062
  Sheep anti-DIG	Roche	1 333 089
  Sheep anti-DIG-POD	Roche	1 207 733
  Mouse anti-BIO	Roche	1 297 597
  Goat anti-BIO	Rockland Immuno	600-101-098
  Streptavidin-HRP	Jackson Immuno	016-030-084
  Mouse anti-FITC	Roche	1 426 320
  Goat anti-FITC	Rockland Immuno	600-101-096
  Rabbit anti-FITC	Molecular Probes	A-889
  Rabbit anti-FITC-HRP	Molecular Probes	A-21253
  Rabbit anti-DNP	Molecular Probes	A-6430

• PBT+ Blocking Reagent= Use the Western Blocking Reagent from Roche #1 921 673 (100 ml) as a 5x stock, mixing it with PBT on the day of use (it is sold as a 10x stock for western blots).

• Complete information about all the Alexa fluor conjugates can be found in the Molecular Probes catalog or web site.

Mounting media

• An inexpensive and simple mounting media= 90% glycerol / 100 mM Tris pH 8.0. Combine 1 ml of 1 M Tris pH 8.0 and 9 ml glycerol, and mix well. This medium will not protect against photobleaching, but performs well for most experiments.

• DABCO / Tris / Glycerol (DTG)=
  • 2.5% w/v DABCO, e.g. Sigma #D-2522
    50 mM Tris pH 8.0
    90% glycerol
    
  • For 50 ml, use 1.25 g DABCO, 45 ml glycerol, 2.5 ml 1 M Tris pH 8.0, and 2.5 ml ddH₂O.
  • Combine components, dissolve DABCO by warming to 70° C., mix, aliquot and store at -20° C.
  • Protects well against photobleaching.

• 'Prolong' from Molecular Probes is extremely effective against photobleaching and it hardens for semi-permanent storage at -20° C.