|
X-PHOS/NBT STAINING OF WHOLE MOUNTS See above protocol for X-gal staining of whole mounts and perform steps 1 and 2. 3. Heat tissue for at least 30' at 650C.
For staining of embryonic chick diencephalon (one of the areas of the brain with the highest background), this step was increased to 1.5 hours. It may be that even longer heat treatment could benefit specific staining in areas with high background.
4. Incubate tissue in X-Phos/NBT Detection Buffer for 15' at room temperature.
XPhos/NBT Detection Buffer (Buffer 3 as described for Genius kit by Boehringer-Mannheim)
- 100 mM Tris-HCl, pH 9.5
- 100 mM NaCl
- 50 mM MgCl2
Store at room temperature. Tends to precipitate over several weeks. This does not seem to noticeably affect the staining. 5. Incubate in X-Phos/NBT Reaction Solution for 1 to several hours at room temperature. Cover the reaction with foil to reduce background.
X-Phos/NBT Reaction Solution
- 50 ul of 100X X-Phos stock
- 100 ul 50X NBT stock
- 50 ul of 100X levamisole stock, optional
- in 5 ml X-Phos Detection Buffer
- Mix X-Phos, NBT, and levamisole with the Detection Buffer immediately before using.
Stocks
- X-Phos (100X):10mg/ml 5-bromo-4-chloro-3-indolyl-phosphate (also referred to as BCIP) in H20. Store in the dark as aliquots at -200C. Can be frozen and thawed several times.
- NBT (50X):50 mg/ml nitro blue tetrazolium in 70% dimethylformamide, 30% H2O. Store at -200C in a glass container covered with foil. Does not freeze at this temperature.
- Levamisole (100X): 50 mM in H20. Store at -200C.
- PMS (100X): 2 mg/ml phenazine methosulfate in H20. Use immediately.
6. Rinse in 20mM EDTA in PBS for 2-4 hours.
Tissue can be stored in the dark at 40C in PBS + EDTA or 30% sucrose in PBS + EDTA + 0.05% sodium azide for many months, although the background clearly increases over time. Tissue can then be sectioned as above for X-gal stained whole mounts.
Notes
- 0.5% glutaraldehyde decreased PLAP activity in embryonic chick brain cells stained in whole mounts, but not in chicken embryo fibroblasts cultured in vitro. Fixation of chick brain whole mounts thus is typically done in 4% paraformaldehyde in PBS for 2-4 hours at 4OC. In areas where background alkaline phosphatase activity is a problem, increasing the time in 4% paraformaldehyde, even up to several days, can decrease endogenous background without significantly decreasing PLAP activity.
- Chick retinas and cerebella have been kept in PBS at 4OC for at least one month after fixation and rinsing in PBS with no appreciable loss of signal in X-Phos staining.
- Background staining can be due to endogenous alkaline phosphatase activities or reduction of NBT from other sources (e.g. NADPH). It is also enhanced by light. The most effective inhibitor is heat. To reduce background further, one or more of the following inhibitors can be tried in addition to heat (they are added to the reaction mix): 0.5 mM levamisole (L[-]-2,3,5,6-tetrahydro-6-phenylimidazo{2,1-b}thiazole), 2 mM mercuric chloride, 5 mM L-leucyl-glycyl-glycine, 1 mM EDTA, 1 mM L-phenylalanine-glycyl-glycine, 0.2 M lysine HCl or 0.3 mM sodium arsenate (Zoellner and Hunter, 1989). We found that levamisole was the second most useful treatment (after heat) in reducing the background staining in brains, although it also reduced PLAP staining slightly in some cases.
- If background staining is not a problem, the reaction can be continued for up to 2 days.
X-PHOS/NBT STAINING OF SECTIONS
Cryostat sections can be stained for PLAP activity. Follow the protocol listed above for X-gal through step 5.
6.Transfer slides to preheated PBS at 65OC and heat for 30 minutes.
7. Rinse slides in room temperature PBS for 5 minutes.
8. Rinse slides in X-Phos/NBT Detection Buffer for 10 minutes.
9. Stain slides in X-Phos/NBT Reaction Mix for 1 to 12 hours at room temperature. Cover with foil during and after staining. The time in the reaction buffer will depend on the level of PLAP expression and endogenous background. If background is low, staining can continue for 2 days.
10. Rinse slides in PBS + 20 mM EDTA, 3x 10 minutes.
11. Mount in Gelvatol (+ 20 mM EDTA if desired).
Storing slides at -80oC helped prevent background staining from increasing.
Notes
- 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 6 above.
- Processing cultured cells grown on glass through the procedure for paraffin embedding prior to staining preserved enough PLAP activity that positive cells were visible. If paraffin sections are desirable, it is worth trying pilot experiments using the tissue of interest, varying the fixation, and minimizing the times in organic solvents.
X-gal AND X-PHOS/NBT STAINING
ß-gal and PLAP can be detected in the same tissue and even in the same cell. In order to process tissue for both activities, staining with X-gal must be done first as the lacZ-encoded enzyme is destroyed by the heating step used to reduce endogenous alkaline phosphatase activities. To combine the protocols, proceed through the X-gal reaction as described above for either whole mounts or sections. You may wish to stop and examine the results before moving to PLAP staining. The indigo product of X-gal can be easier to detect prior to carrying out the X-Phos reaction as background alkaline phosphatase staining can obscure it, particularly in whole mounts. Rinse the tissue very well with PBS prior to PLAP staining as residual ß-gal activity in the presence of X-gal and NBT can enable ß-gal+, PLAP- cells to turn purple. Follow the above protocols for PLAP staining.
A COMPARISON OF ALTERNATIVE SUBSTRATES FOR ß-gal AND PLAP
As described above, there are a number of alternative substrates for both enzymes. The following discussion concerns these substrates and summarizes our experiences with them relative to the those described above.
ß-gal SUBSTRATES:
X-gal is a good choice as a precipitable substrate with iron as an electron acceptor. It gives a bright blue color, although the reaction is quite slow, even at 37OC. NBT added to X-gal gives an intense purple color which develops much more rapidly. PMS will increase this reaction rate even further. Tetrazolium red makes the product look greenish-blue, but increases background, making the staining more equivocal. Other modified indolyl-based compounds can be used if alternative colored precipitates are desired. Salmon-gal (Biosynth International) results in a light orange/pink precipitate which develops slowly. Adding tetrazolium salts tends to increase the background. Magenta-gal (Biosynth International) gives a light pinkish purple precipitate. Green-gal (Biosynth International) did not give discernible precipitates in our hands, except in the presence of NBT, which gave a purple precipitate.
PLAP substrates
X-Phos in the absence of NBT gives a bright blue precipitate which develops slowly over time. As a result, there may be some diffusion away from the site of enzyme activity. Iron does not work as an electron acceptor for the indolyl-based compounds at high pH, and results in large amounts of floating precipitate. Adding NBT results in a deep purple precipitate which develops more quickly. Adding PMS increases the rate of the reaction (in the presence of NBT) without adding background by quantitatively reducing the tetrazolium salt. Other modified indolyl-based compounds can be used if alternative colored precipitates are desired. Magenta-phos (Biosynth International) gives a pinkish-purple precipitate which develops at intermediate rates. The color can be distinguished from the purple color derived from X-Phos + NBT. Adding tetrazolium red or blue makes the product look more purple (less pink). Salmon-phos (Biosynth International) results in a light orange-pink precipitate which develops slowly. Adding tetrazolium red or blue gives a purple precipitate similar to XP + NBT. The Vector Red substrate kit (Vector Laboratories) results in staining that is highly variable between experiments; the precipitate can be intensely red but is usually light pink. The substrate is unstable, and begins to form floating precipitates after 20-30 minutes. Addition of tetrazolium salts is not helpful, and PMS acts as an inhibitor in this system. The substrate affords high background levels to intact tissues and thus is not useful for staining whole mounts. The blue substrate kit (Vector Laboratories) affords a very pretty peacock blue precipitate, but has the same problems as the red substrate kit, i.e. unstable substrate and high background to intact tissues. Staining is reproducible and intense, however. Adding tetrazolium salts such as NBT increases the background and results in copious amounts of non-localized precipitate.
Legend for Figure 1. Production of indigo from X-gal or X-Phos.
A. Production of the stable blue precipitate from substituted indoles proceeds as shown. Two molecules of either X-Phos or X-gal generate 2 molecules of indoxyl which then form the indicated dimer. The "X" moiety (5-bromo-4-chloro-indolyl) in both X-gal and X-Phos is the same and thus once cleavage by the respective enzyme has occurred, the dimerization and oxidation reactions lead to the same halogenated indigo compound.
B. Nitroblue tetrazolium (NBT) is an example of a tetrazolium salt. These are a rather unstable class of compounds which precipitate when reduced to form highly colored compounds. In this case, NBT is reduced by the hydride produced by dimerization of the two indoxyls that result from cleavage of either X-gal or X-Phos (shown in (A) above). When reduced, NBT forms formazan, a dark purple precipitate.
REFERENCES
- *Altman, F.P. 1972. Quantitative dehydrogenase histochemistry with special reference to the pentose shunt dehydrogenases. Progr. Histochem. Cytochem. 4:225.
- Berger, J., A.D. Howard, L. Gerber, B.R. Cullen, and S. Udenfriend. 1987. Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc. Natl. Acad. Sci. USA 84:4885.
- Beckwith, J.R. 1980. Lac: The genetic system. In The Operon (ed. J.H. Miller and W.S. Reznikoff). Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
- Bonnerot, C., Rocancourt, D., Briand, P., Grimber, G., and Nicolas, J.-F. 1987. A ß-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc. Natl. Acad. Sci. USA 84:6795.
- Cotson, S. and S.J. Holt. 1958. Studies in enzyme cytochemistry IV. Kinetics of aerial oxidation of indoxyl and some of its halogen derivatives. Proc. Royal Soc. (London) 148B:506.
- Fekete, D.M., J. Perez-Miguelsanz, E.F. Ryder, and C.L. Cepko. Clonal analysis in the chicken retina reveals tangential dispersion of clonally related cells. Dev. Biol. In press, 1994.
- *Fields-Berry, S.C., A.L. Halliday, and C.L. Cepko. 1992. A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. Proc. Natl. Acad. Sci. USA. 89:693.
-
*Fire, A. 1992. Histochemical techniques for locating E. coli ß-galactosidase activity in transgenic organisms. Genet. Anal. Tech. Appl. 9:151.
- Halliday, A.L., and C. Cepko. 1992. Generation and migration of cells in the developing striatum. Neuron 9:15.
- Henthorn, P., P. Zervos, M. Raducha, H. Harris, and T. Kadesch. 1988. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression. Proc. Natl. Acad. Sci. USA 85:6342.
- Holt, S.J., and D.G. O'Sullivan. 1958. Studies in enzyme cytochemistry I. Principles of cytochemical staining methods. Proc. Royal Soc. (London) 148B:465.
- Holt, S.J., and P.W. Sadler. 1958. Studies in enzyme cytochemistry III. Relationships between solubility, molecular association and structure in indigoid dyes. Proc. Royal Soc. (London) 148B:495.
- Hugon, J., and M. Borgers. 1966. A direct lead method for the electron microscopic visualization of alkaline phosphatase activity. J. Histochem. and Cytochem. 14:429.
- Kam, W., E. Clauser, Y.S. Kim, Y.W. Kan, and W.J. Rutter. 1985. Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA. Proc. Natl. Acad. Sci. USA 82:8715.
- Krasnow, M.A., S. Cumberledge, G. Manning, L.A. Herzenberg, and G.P. Nolan. 1991. Whole animal cell sorting of Drosophila embryos. Science 251:81.
- Lin, S., S. Yang, and N. Hopkins. 1994. LacZ expression in germline transgenic zebrafish can be detected in living embryos. Dev. Biol. 161:77.
- Lojda, Z. 1970. Indigogenic methods for glycosidases. Histochemie 22:347.
- McCaman, R.E., and E. Robins. 1959. Quantitative biochemical studies of Wallerian degeneration in the peripheral and central nervous systems-II. J. Neurochemistry 5:32.
- McComb, R.B., G.N. Bowers, Jr., and S. Posen. 1979. Alkaline Phosphatase. Plenum Press, New York.
- Miller, J. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
- Nirenberg, S., and C. Cepko. 1993. Targeted ablation of diverse cell classes in the nervous system in vivo. J. Neuroscience 13:3238.
- Pearse, A.G. 1954. Azo dye methods in enzyme histochemistry. In International Review of Cytology vol. III (ed. G.H. Bourne, and J.F. Danielli) Academic Press, New York.
- Pearson, B., P.L. Wolf, and J. Vazquez. 1963. A comparative study of a series of new indolyl compounds to localize ß-galactosidase in tissues. Lab Investig. 12:1249.
- Rentrop, M., B. Knapp, H. Winter, and J. Schweizer. 1986. Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pre-treatment conditions.Histochem. J. 18:271.
- Rodriguez, J., and F. Deinhardt. 1960. Preparation of a semipermanent mounting medium for fluorescent antibody studies. Virology 12:316.
- Rosenberg, W.S., X.O. Breakefield, C. DeAntonio, and O. Isacson. 1992. Authentic and artifactual detection of the E. coli laxZ gene product in the rat brain by histochemical methods. Brain Research 16:311.
- Snyder, E.Y., D.L. Deitcher, C. Walsh, S. Arnold-Aldea, E.A. Hartwieg, and C.L. Cepko. 1992. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 68:33.
- Vaysse, P.J.-J., and J.E Goldman. 1990. A clonal analysis of glial lineages in neonatal forebrain development in vitro. Neuron 5:227.
- Wallenfels, K., and R. Weil. 1972. In The Enzymes. (ed. P.D. Boyer). 3rd edition. 7:617. Academic Press, New York.
- Zoellner, H.F., and Hunter, N. 1989. Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase. J. Histochem. and Cytochem. 37(12):
共2页: 上一页 [1] 2 下一页
上一篇：激光显微切割 Laser Microdissection protocols 下一篇：新鲜组织石蜡快速处理 Rapid Processing of Fresh Tissue into Paraffin
|
|
激光显微切割 Laser Micro