(X-gal) (Eurogentec, Seraing, Belgium) sold in powder form. Both the powder and stock solution (40 mg/mL) prepared in dimethyl sulfoxide (DMSO; Sigma) must be kept at -20°C.
M.
a stock solution of 20% in PBS and store at -20°C.
perfusion.
France) for perfusion of animals and fixation of tissues by perfusion.
France) to section the tissues (20–40 μm thickness).
sections from one solution to another. Obtain from any art equipment supplier.
sections.
ferrocyanide (Sigma): prepare 0.2 M stock solutions of each.
70%, 95%, and 100% of ethanol).
alcohol (all from Sigma) for delipidation of whole newborn brains and for small blocks of tissue from adult brains.
(glycerol/PBS, 1/3 vol/vol) for mounting slides.
benzyl alcohol are irritants.
and emission filters for fluorescein.
β-gal can be revealed by several means: 1. Histochemical X-gal revelation, whether on sections or carried out in toto, requires intracardial perfusion of the anesthetized animals using a peristaltic pump. First, tissues are fixed by perfusing 2% PFA (in PBS), then fixation is continued by leaving tissues blocks overnight in the same solution. Tissues are then vibratome sectioned, and sections are incubated in a 0.4 mg/mL Xgal solution for 2 to 4 hours (30°C). It is important to precede the fixation by perfusion with saline containing 10 U of heparin to help remove blood and blood cells from the vessels. The fixative (2% PFA) can contain EGTA 1.25 mM and MgCl2 2 mM, which improves the X-gal reaction. To make the reaction mixture, the stock solution of X-gal (40 mg/mL in DMSO) is diluted to 0.8 to 1 mg/mL PBS containing: 0.1% Tween 20, 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, and 2 mM MgCl2. 2. Immunocytochemical revelation also requires fixed vibratome sections mounted on cromallun–gelatin-coated slides. Polyclonal anti-β-gal monoclonal anti-GFAP and monoclonal anti-NeuN antibodies are used in our experiments. Monoclonal as well as polyclonal antibodies can be labeled using appropriate labeling kits from Amersham Pharmacia Biotech (Piscataway, NJ, USA). We used Cy3.5 (Fluorolinf-ab) according to the manufacturer’s instructions to label monoclonal antibodies. Primary antibodies are diluted to the concentrations recommended by each manufacturer in PBS 0.1 M (containing gelatin 0.2%, TritonX-100 0.3%, 3% normal goat serum). Anti-β-gal is revealed using fluorescein coupled antirabbit antibody. Sections are protected from light to avoid fading of the fluorescence and mounted with glycerol/PBS (1/3 vol/vol) or Vectashield and examined under a fluorescence microscope. Luciferase antibodies can also be used to follow transgene expression in a double labeling protocol. However, there are currently some problems with obtaining good luciferase antibodies for in vivo work (see Discussion). 3. Fluorescein digalactoside (FDG) is another substrate for β-gal. Hydrolysis of FDG by β-gal results in the liberation of both a monogalactoside and fluorescein. This second product is easily detectable and theoretically makes this method very sensitive. Its main limitation for us is that it is not suitable for fixed tissues, so it is difficult to obtain good morphology in brain preparations. Moreover, on unfixed tissue, as cells die, the fluorescein product diffuses out. Thus, the revelation procedure must be very fast. Also, it is not possible to perform double staining to identify cell types. So this method, which has the theoretical advantage of higher sensitivity than X-gal, is in fact rather limited for in vivo studies. 4. In toto X-gal revelation require intracardial perfusion of the anesthetized animals using a peristaltic pump. Fixation and postfixation are performed as for vibratome sections, but organs are treated as whole mounts. Incubate tissues in a 0.4 mg/mL X-gal solution for 2 to 4 hours (30°C), rinse in 0.1 M PBS (2 × 5 min) and transfer in series of ethyl alcohol under mild agitation: 70% (2 × 2 h), 95% (overnight and another bath of 1 h), 100% (2 × 2 h). Dehydration times can be adapted depending on the tissue size. To ensure thorough dehydration, one can leave the tissue in the second 100% alcohol bath overnight. Put dehydrated tissues into xylene (2 × 2 h in glass), then transfer them into benzyl benzoate–benzyl alcohol (2/1 vol/vol) in a glass container until clarification. Note: Use gloves and glass containers at all steps involving benzyl benzoate and benzyl alcohol. These agents are irritants and also dissolve plastic.
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