2) Fix whole adults in 0.5ml of fixative-saturated heptane for 20 minutes at room temperature. Cover the wells with a sideglass to prevent heptane from evaporating.
3) Remove adults from heptane and place on a piece of blotting paper for a few seconds to evaporate heptane. Place in a drop of postfix solution in a shallow depression slide. Remove body from the head and discard.
4) Using two tungsten needles, dissect retina from head as follows. First, cut along the edge of the lens and remove retina + cornea from the rest of the head. In most cases a thin pigmented layer of cells located between the retina and the lamina will come off the retina. Since in many lacZ lines this layer stains heavily and masks the expression in the retina, it needs to be removed by scratching with a fine needle. Then, remove the lens from the retina by inserting the needle between the retina and the cornea. Removal of the cornea is not absolutely necessary for staining, but it accelerates bleaching of the pigments and will also make the preparation hydrophilic. Transfer the dissected retina to postfix solution in a microtiter plate and fix for an additional 10 minutes.
5) Remove the postfix solution using a drawn out Pasteur pipet and rinse once with PBS, and then replace the PBS with staining solution without X-gal (i.e., Fe/NaP or Fe/CP). Leave at room temperature about 5 minutes.
6) Remove liquid and replace with staining solution. Incubate 2 hours to overnight at room temperature or 37°C.
7) Remove stianing solution. Rinse retina once in 70% ethanol and once in 100% ethanol. This step helps to remove X-gal crystals that are adhering to the sample.
8) Remove ethanol and add 50% glycerol/PBS. Take retina out into a drop of 50% glycerol in a depression slide and remove X-gal crystals with a fine needle. Mount in 50% glycerol using one #1 coverslip on one slide.
Staining with Fe/NaP pH7.2 and higher temperature (37°C) results in stronger staining, presumably because the conditions are closer to the optimum for the enzymatic activity. For Rh4/lacZ fusion gene transformants, using Fe/NaP overnight at room temperature allows you to stain overnight, rather than stopping the reaction after a short incubation period. Staining at 37°C will increase the size of reaction product crystals and will make identification of stained cells more difficult.
Fixative-saturated Heptane (Prepare fresh solution every day)
0.1M Na cacodylate buffer pH7.3 1ml
50% gluteraldehyde 1ml
(EM grade, Fluka #49631)
heptane 2ml
Shake well and let the phases separate. Use the heptane (upper) phase for fixation. Fixative (lower) phase can be reused by adding heptane to 2ml.
Postfix Solution
0.1M cacodylate buffer pH7.3 2ml
water 2ml
50% gluteraldehyde 80µl (1% final concentration)
(EM grade, Fluka #49631)
10% Triton X-100 20µl (0.05% final concentration)
Fe/NaP (pH7.2)
0.2M Na2HPO4 1.8ml
0.2M NaH2PO4 0.7ml
5M NaCl 1.5ml
1M MgCl2 50 µl
50mM K3(Fe(CN)6) 3.05ml
50mM K4(Fe(CN)6) 3.05ml
H2O to 50ml
Store in the dark at room temperature.
Fe/CP (pH8)
50mM K3(Fe(CN)6) 5ml
50mM K4(Fe(CN)6) 5ml
CP pH8 40ml
Store in dark at room temperature.
CP (pH8)
0.1M citric acid 27.5ml
Na2HPO4 27.61g
H2O to 1 liter
Staining Solution
Warm up Fe/NaP or Fe/CP to 37°C. Add 1/30 volume of X-gal
(5-bromo-4-chloro-3-indolyl- -D-galactopyranoside) solution (8% in DMF
(N,N-dimethylformamide), store at -20°C).