Reagents and Recipes

Fixation:

• 50% bleach= 1:1 mix of ddH₂O:unscented supermarket bleach that is not too cheap

• 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%
     • I think the 37% formaldehyde doesn't work as well after some months of the bottles being opened, but countless millions of embryos have been stained well using it.
  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 my current working fix recipe

Embryo pre-treatment and hybridization:

• PBT= 1x PBS, 0.1% Tween-20
  • you can 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 of PBT at 4° C. if you don't use it up within a few days

• PBT+ 5% formaldehyde=
  • 1:1 mix of PBT:10% formaldehyde OR
    6:1 mix of PBT:37% formaldehyde
  • make the amount you need on the day of your experiment, it doesn't keep well: once I ruined the embryos with some that was a couple of days old

• 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
  • find the best final concentration at which to use your stock: 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 your stock by performing parallel experiments using different concentrations for some set time. Too low, your signal will be reduced; too high, the embryos will fall apart during hybridization or look mushy, fuzzy, fluffy, or just plain bad; and just right, you'll get great signal on firm and beautiful embryo bodies.

• 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
    
  • good molecular biology grade formamide doesn't need to be de-ionized or filtered before use
  • you can use regular autoclaved ddH₂O to make up this solution
  • some protocols call for 50 µg/ml tRNA as additional carrier and competitor (my probe stocks already contain some tRNA)
  • adjusting the pH is not necessary unless you are planning to use a higher hybridization temperature (>55° C.). The solution will become more basic at the higher temperatures, so you compensate by lowering its pH to begin with. The protocols I've seen call for adjusting the pH down to between 4.5-6.5, either with 1 M citric acid or 1 N HCl, for use at hybridization temperatures between 60°-70° C. I'm still hybridizing at 55° C. and don't bother to adjust it.
  • salmon testes DNA stock solution can be prepared simply by autoclaving it for 20 minutes after having stirred it into solution, instead of sonicating and boiling it.

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
  RNase Inhibitor	Roche	799 017
  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= you have to make it up yourself, using:
  • 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 you need it (it is sold as a 10x stock for westerns). I've tried other traditional blocking reagents with little success. Supermarket non-fat dry milk destroys the signal, and, strangely enough, BSA containing buffers actually increase the background relative to using no blocking substance at all. I don't know why I've had such bad luck with using BSA in this protocol. Since starting to use this blocking reagent from Roche, background problems associated with some of the primary antibodies listed above have vanished and in some cases the embryo can't be seen at all in images whose fluorescent signal is saturated.

• Finally, complete information about all the Alexa fluor conjugates can be found in the Molecular Probes catalog or web site. Feel free to substitute in your own favorite fluorescent conjugates. We are collaborating with Molecular Probes and they have provided many samples of reagents to help develop this protocol, so naturally our bias is toward the Alexa dyes; however, in my opinion, they really are superior to the spectrally equivalent CyN dyes or FITC, TRITC, etc. in terms of brightness, and brightness is what you need when you're doing non-enzymatic immunofluorescence.

Mounting media:

• A very cheap and easy-to-make 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. That's it. It won't protect against photobleaching, but works fine 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

• I've heard that 'Prolong' from Molecular Probes is extremely effective against photobleaching. Also, it hardens for semi-permanent storage, but according to some, that hardening may ultimately warp the embryos.