DETECTION OF ß-GALACTOSIDASE AND ALKALINE PHOSPHATASE ACTIVITIES IN TISSUE

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DETECTION OF ß-GALACTOSIDASE

The E. coli lacZ gene encoding ß-galactosidase (ß-gal) is the classical histochemical reporter gene (Beckwith, 1980). It can be detected using a variety of substrates, all of which have galactose linked through a ß-D-glycosidic linkage to a moiety whose properties change upon liberation from galactose (Wallenfels and Weil, 1972). Several substrates yield colored or fluorescent soluble products which are useful when quantifying ß-gal activity (McCaman and Robins, 1959, Miller 1972) or visualizing transduced cells live in vivo (Krasnow et al. 1991, Nirenberg and Cepko, 1993, Lin et al. 1994). The fluorescent products can even be used to kill cells in vivo (Nirenberg and Cepko, 1993). However, for localization of cells containing transduced lacZ, chromogenic substrates that yield a precipitated product are desirable (Holt and O'Sullivan, 1958, Pearse 1954, Pearson et al 1963). The most common such substrate is an indole derivative, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal, Holt and Sadler, 1958).

When ß-gal cleaves the glycosidic linkage in X-gal, a soluble, colorless indoxyl monomer is produced. Subsequently, 2 of the liberated indoxyl moieties form a dimer which is nonenzymatically oxidized . The resultant halogenated indigo is a very stable and insoluble blue compound (Holt and Sadler, 1958). The dimerization and oxidation reactions require transfer of an electron , which is facilitated by electron acceptors of the proper redox potential (Cotson and Holt, 1958). The ferric and ferrous ions included in most X-gal reaction buffers provide this function (Lojda, 1970). Tetrazolium salts, which can serve as the final electron acceptors, also can be added, and precipitate when reduced to form colored formazan compounds (Altman, 1972). Phenazine methosulfate (PMS) can further increase this reaction rate by quantitatively reducing tetrazolium salts (Altman, 1972). Alternative staining protocols that yield different colored products have been developed based upon these considerations and will be discussed at the end.

X-GAL STAINING OF INTACT TISSUE (WHOLE MOUNTS)

1. Dissect embryos or small pieces of tissue (e.g. the size of a retina from an adult rat works well with this procedure) into PBS containing 2mM MgCl2 (PBS + Mg2+) on ice.
   PBS(10X)

   80 g NaCl

   2 g KCl

   11.5g Na2HPO4

   2 g KH2PO4

   in 1 liter H20

   Fix in 0.5% glutaraldehyde in PBS + Mg2+ or 2.0%- 4.0% paraformaldehyde in PBS + Mg2+ on ice for 30' to several hours. The amount of time should be determined empirically. Generally, it is wise to minimize time in fixative as the lacZ encoded enzyme can be overfixed. Fixation with 0.5% glutaraldehyde gives superior staining relative to paraformaldehyde fixation, but can also preserve endogenous enzyme activity to the point where the signal from endogenous activity is confounding.
   0.5% glutaraldehyde

   Make from a 25.0% stock immediately before use. You can buy a 25% solution (Sigma) and freeze-thaw it many times.
   4.0% paraformaldyde

   4 g paraformaldehyde

   2 mM MgCl2 (0.2 ml of a 1 M stock)

   1.25 mM EGTA (0.25 ml of a .5M EGTA stock, pH 8.0)

   in 100 ml PBS, pH 7.2-7.4 Heat about 80ml H₂O to 60⁰C and add paraformaldehyde; add 1-2 drops of 10M NaOH to get paraformaldehyde in solution. Cool to room temperature, add 10 ml 10X PBS, adjust pH with HCl, add MgCl2 and EGTA, and make up to 100ml with H2O. The solution can be stored at 4⁰C 1-2 weeks.

   Rinse in many changes of PBS. Residual fixative can inhibit the enzyme. Rinsing overnight is fine, but waiting for several days at this step may decrease ß-gal activity, and paraformaldehyde fixed tissue can become "unfixed".

   Dilute the X-gal stock into X-gal Reaction Buffer and incubate with the tissue 2-4 hours at 37⁰C.

   X-gal Reaction Buffer

   • 35 mM potassium ferrocyanide (can vary from 5-35 mM)
   • 35 mM potassium ferricyanide (can vary from 5-35 mM)
   • 2 mM MgCl2
   • 0.02% Nonidet P-40 (NP-40) (diluted from 10% stock solution)
   • 0.01% Na deoxycholate (diluted from 10% stock solution) in PBS

   X-gal reaction buffer can be stored for at least one year at room temperature in foil-covered container.

   • X-gal stock(40X) 40 mg/ml X-gal in dimethylformamide
   • Store at -20⁰C in foil covered glass container.

   Rinse many times in PBS until the solution no longer turns yellow. This usually takes about 5 changes. O/N rinse is fine.

   View under bright field optics for optimal detection.

   Notes

   1. Although we have seen reduced activity of ß-gal after treatment with organic solvents, lyophilization followed by permeabilization in acetone has been used successfully in place of aldehyde fixation for X-gal staining in C. elegans (Fire, 1992).
   2. The amount of ferricyanide and ferrocyanide can be varied. A more rapid precipitation is achieved with the higher concentration, but the higher concentration can lead to a greenish precipitate (Prussian Green) in tissue upon prolonged staining.
   3. The ferricyanide and ferrocyanide can form a blue precipitate (Prussian blue) upon reaction with free ferric ion. Do not use metal forceps to manipulate the tissue while it is in the X-gal detection buffer.
   4. iv. All tissues have endogenous, lysozomal ß-gal. Its pH optimum is very low, and thus it is not very active in the pH 7.4 buffer used here. However, some tissues also have a cytosolic form of ß-gal which can show enough activity to be confounding. The variables that affect background staining include the fixative type, length of time in fixative, pH of the buffer, and amount of time in the staining solution (Rosenberg et al 1992). If after varying these parameters, background is still a problem, one can detect the lacZ enzyme by immunohistochemistry. Monoclonal and polyclonal antibodies are available (e.g. from 5 prime, 3 prime, Boehringer-Mannheim and Cappel).
   5. Tris buffer was tried in place of PBS in the reaction buffer, with no success.
   6. The detergents in the X-gal reaction buffer need not be included, but usually do not reduce staining, and for some tissues they increase staining.
   7. The indigo product is soluble in organic solvents and thus one should minimize exposure to such solvents after formation of the product. Nonetheless, fixation with glutaraldehyde (but not formaldehyde) allowed preservation of enough indigo to make it through the various solvents required for embedding for electron microscopy (EM) (Snyder et al. 1992).
   8. Double staining of cells for ß-gal with X-gal and a cellular antigen using antibodies is difficult, though possible (Snyder et al 1992, Vaysse and Goldman, 1990). The indigo product of X-gal absorbs in the wavelengths emitted by the standard fluorescent-conjugated antibodies and is so dark that it can obscure the products from either horseradish peroxidase or alkaline phosphatase-conjugated antibodies. However, by carefully controlling the reaction time in X-gal so that only a small amount of indigo is produced (Vaysse and Goldman, 1990), by having ß-gal localized to the nucleus when the cellular antigen is non-nuclear (Bonnerot et al 1987), or by using antibodies to detect both ß-gal and the cellular antigen, one can overcome these problems.
   9. If nitroblue tetrazolium (NBT) salt is added to the X-gal reaction in place of iron, a purple precipitate will result. This can be a faster and more sensitive reaction than X-gal alone. A stock solution of NBT can be prepared by adding 50 mg NBT per ml of 70% dimetnylformamide and stored at -20o C. Final working concentrations are 0.25 - 1.0 mg/ml. Phenazine methosulfate (PMS) can be added in conjunction with NBT to increase the reaction rate further. Prepare a 100X stock of 2 mg/ml in H2O and use immediately. PMS is very unstable.
   10. To aid in identification of stained cells, the tissue can be processed for cryostat sections as discussed below. In addition, staining cryostat sections may give higher signal than whole mounts so if you are not sure that the whole mount procedure gave the maximal staining, stain sections as below. Alternatively, for simply visualizing the stained cells produced during staining of whole mounts, paraffin sections can be made (paraffin embedding destroys ß-gal activity so there is no point in staining paraffin sections). Embed in paraffin using minimum necessary times for the tissue of interest as the solvents can partially dissolve indigo. For glutaraldehyde-fixed mouse retina, which is approximately 250 microns thick, the following procedure was used. Dehydrate through graded ethanols (50%, 70%, 95%, 100%, 100%) for 20 min each. Clear in xylene, 2 x 15 min. Infiltrate with 1:1 mix of xylene and paraffin, 65^OC for 30 min. Paraffin, 2 x 15 min. Embed in paraffin.

   X-GAL STAINING OF TISSUE SECTIONS

   The procedure for staining tissue sections is very similar to the protocol for intact tissue. Section staining should be used when a whole mount cannot be used due to the size of the tissue.

   1. Fix tissue in fixatives listed above using perfusion if possible and follow with immersion in fixative at 4^OC for several hours. Rinse briefly in PBS, then sink in 30% sucrose in PBS + Mg2+at 4^OC.

      Fixation times will vary with the size and nature of the tissue. For embryonic chick brains of E10 or older and for postnatal rat or mouse brains we incubate up to 8 hours in fixative. Perfusion may not be necessary for all tissues and shorter fixation times may be preferable as X-gal staining may be decreased by lengthy fixation.

   2. Embed tissue in OCT (Miles) or gelatin/sucrose mounting medium and freeze on metal chucks cooled in liquid N2.

      Gelatin/sucrose embedding gives better frozen sections for embryonic tissue than does OCT.

      Gelatin/sucrose embedding solution
      • 7.5% gelatin (porcine skin, Sigma)
      • 15% sucrose
      • 0.05% sodium azide
      • in 1X PBS
        
      Dissolve gradually at 60oC, with stirring. The medium solidifies at room temperature to a transparent gel. Store at room temperature. Liquefy in microwave with frequent swirling before embedding samples.

   3. Cut cryostat sections and mount on silane-coated (Rentrop et al., 1986) or gelatin-coated slides; air-dry O/N. Sections up to 90 m m thick (the thickest we have tried) have been successfully stained.
      • Gelatin Solution for subbing slides
      • 2 g gelatin
      • 0.1 g chromium potassium sulfate (chrome alum)
      • in 200 ml H20

      Heat H20 to 60oC. Dissolve chrome alum, then gradually dissolve gelatin. Filter before use. Can increase or decrease the percentage of gelatin. Load slides in racks, dip quickly, and air-dry overnight.

   4. Fix sections to slides in 4% paraformaldehyde for 10-15 minutes at 4^OC.

   5. Rinse slides in PBS + Mg2+ twice, for 10 minutes each, at 4^OC.

   6. Stain slides in X-gal Reaction Buffer for 1-24 hours at 37^OC.

   7. Rinse slides in PBS 3 times, for 10 minutes each, or until solution is no longer yellow. Slides can be left in PBS O/N.

   8. Coverslip in gelvatol.

   GELVATOL PREPARATION

   We have revised the protocol published by Rodriguez and Deinhardt (1960).

   1. Take 200 ml of 0.01M KH2PO4 (about pH 5.0) and add enough 0.01 M Na2HPO4 to bring the pH up to 7.2.
   2. Take 250 ml of the 0.01M KH2PO4/Na2HPO4 and add 2.05 g NaCL to give a 0.14M NaCL concentration.
   3. Dissolve 62.5 g Gelvatol (Air Products) in the 250 ml of 0.01 M KH2PO4/Na2HPO4/.14 M NaCL. Stir on magnetic stirrer in warm room for a few hours.
   4. Add glycerol in an amount equal to one-half the total volume of the Gelvatol buffered saline solution and stir overnight at room temperature.
   5. Centrifuge the Gelvatol solution at 12,000 rpm for 15 min. in 30 ml Corex tubes in Beckman J2-21 centrifuge at room temperature to remove undissolved particles.
   6. Pipette the supernatant into small screw cap bottles. Check pH of Gelvatol solution. It should be between pH 6 and 7.
   7. Store Gelvatol solution at 4^oC. Screw caps on tightly to prevent evaporation. Do not leave Gelvatol uncapped for longer than necessary when working with it.

   Notes

   1. The amount of time for the X-gal reaction will vary according to the concentration of lacZ and the amount of endogenous ß-gal.
   2. If cultured cells are to be stained, fix in 0.5% glutaraldehyde in PBS or in 4.0% paraformaldehyde in PBS for 5' at room temperature and proceed from step 5 above. Fixation for > 5 minutes can lead to decreased enzyme activity.

   STAINING FOR ALKALINE PHOSPHATASE ACTIVITY

   Phosphatase genes are useful reporter genes as several histochemical methods that yield precipitated, highly colored and/or electron dense products have been devised. A chromogenic substrate, 5-bromo-4-chloro-3-indolyl phosphate (X-Phos), which is very similar to X-gal, can be employed for detection and leads to production of a blue precipitate (Figure 1). In addition, the tetrazolium salt NBT, (Altman, 1972) can be used in conjunction with X-Phos as the final electron acceptor for the indoxyl dimerization reaction. When reduced, NBT forms a purple precipitate.

   Of the cloned phosphatases, the human placental alkaline phosphatase gene, PLAP (Kam et al 1985), is perhaps the most useful as it is very heat stable and is resistant to some chemical inhibitors that are active on other endogenous alkaline phosphatases. PLAP has been used as a histochemical reporter fairly recently (Berger et al 1987, Henthorn et al 1988, Fields-Berry et al 1992). It has not been used as widely as lacZ and thus its neutrality needs to be established. LacZ and PLAP have different strengths and weaknesses regarding their distribution and histological detection. PLAP is normally associated with membranes and thus PLAP activity and defines the outer surface of transduced cells, including neuronal processes. We have found PLAP staining in retinal ganglion cell axons several centimeters from cell bodies (Fekete and Cepko, unpublished). However, there are times when neuronal cell bodies are not well defined, which can make it difficult to count cells (Halliday and Cepko, 1992 and Fekete et al, 1994). ß-gal activity visualized by X-gal typically does not fill cells, particularly long processes, but often gives good staining of cell bodies and thus it is easy to count cells. Frequently, X-gal staining is fairly perinuclear and sometimes it is punctate as well. When X-gal precipitate is viewed under the EM, it appears that it localizes to the nuclear envelope and the golgi and endoplasmic reticulum (Snyder et al 1992). For PLAP, there exists a procedure in which lead is precipitated very close to the location of the enzyme (Hugon and Borgers, 1966). The lead precipitate is superior to that of X-gal and thus PLAP is the enzyme of choice when electron microscopy is required.

   There are endogenous alkaline phosphatase activities that may lead to difficulties in detection of transduced PLAP (McComb et al., 1979). In most cases, background activities can be minimized with heat and chemical inhibitors that leave PLAP active (Zoellner and Hunter, 1989). In addition, monoclonal and polyclonal antibodies specific to PLAP can be purchased (Dako, Zymed, Medix, and Accurate Chemical and Scientific Co.) when the background is not surmountable using the various inhibitors.

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