Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

1.4 L BODIPY-TR-14-dUTP (Molecular Probes)

1.4 L TdT (Boehringer Mannheim/Rocher)

100 L

We tested different manufacturer’s TdT samples; Boehringer/Roche was the most reliable and gave the best labeling. The sample includes the enzyme, reaction buffer, and CoCl2.

Tdt: Boehringer/Roche, cat. # 220 582

BODIPY-TR-dUTP: Molecular Probes, cat. # C7618

YOYO-1: molecular probes, cat # Y3601

Equilibration Buffer: Intergen, cat # S7106

Other useful things to control for successful labeling:

1.A short postmortem interval for all pathology cases (ours were less than 6 h on average). You will notice in many European publications the postmortem intervals are 20 h or greater—this cannot produce reliable ISEL because many nuclei will label because of extensive DNA degradation that occurs postmortem. Try to get less than a 12 h postmortem interval if you can.

2.The tissue should be fixed in buffered formalin and kept refrigerated to provide good DNA preservation for ISEL. Note: We saw no difference in labeling with tissue kept in fixative for 6 months, 2 years, or 3 weeks when we initially tested human brain cryosections.

3.Proteinase K digestion is critical—too long and your background signal is increased (and it is impossible to get rid of), too short and you don’t get good access to the DNA in the nucleus. Always do a test run on a few slides, trying different times. Embryonic tissue is more fragile, needs maybe about half the digest time of adult brain. With some fragile tissues, we have found that a brief exposure to Neuropore (Trevigen) offers good permeabilization. Again the time of exposure must be tested (i.e., 5–15 min).

V.IMMUNOCYTOCHEMISTRY FOR APOPTOSISRELATED PROTEINS—HUMAN POSTMORTEM PARAFFIN SECTIONS

A.GAPDH (Monoclonal Antibody—Chemicon)

We and others found that GAPDH (glyceraldehyde 3-phosphate dehydrogenase) plays a role in the apoptotic pathway of some cells. In our partially neuronally differentiated PC12 cells that enter apoptosis after serum and NGF withdrawal, we found that there is an increase in cytoplasmic GAPDH as well as nuclear accumulation of this protein. Blocking GAPDH nuclear accumulation, either by antisense oligonucleotides [22] or with deprenyl-related propargylamines [23],

reduced the incidence of apoptotic nuclei in vitro. It is not yet clear what role GAPDH plays in the nucleus of cells entering apoptosis. We do know that nuclear GAPDH accumulation occurs before the nuclear degradative changes. This protocol works well for adult brain tissue, but we have noted that we get better results if sections are stored cold, and reacted soon after being cut.

Dewax sections according to standard protocol (see ISEL/YOYO for details).

1.Antigen Unmasking Solution (Vector Labs). 400 mL in Pyrex measuring cup. Heat on High power for about 5 min to bring to boil. Add slides in plastic rack and heat at Power setting 8 for 40–60 s to reach boiling, the let slides boil for 1 min only. Let slides cool in buffer for 30–40 min on benchtop.

2.PBS rinse, 1 5 min.

3.Methanol, 20°C, 10 min. Rinse with PBS.

4.RNAse A digest, 100 g/mL in 2 SSC, prewarmed to 37°C, for 3 min only! This step allows antibody access to GAPDH, which is often found in association with RNA in the cell.

5.Ice-cold 0.1 M glycine/0.1 M Tris buffer (pH 7.2) for 5 min to stop enzyme digest and decrease background.

6.PBS rinse, room temperature, 5 min.

7.10% Normal Goat Serum/0.2% Tween 20, 15 min to block.

8.Primary Antibody (1 : 200) in 1% NGS/0.2% Tween 20/0.1 M PBS overnight, 4°C. Individual sections are covered with a parafilm “coverslip.”

9.PBS rinses, room temperature, 4 .

10.Secondary Antibody, goat anti-mouse IgG–Alexa 594 (1:200), in 1% NGS/0.2% Tween 20/0.1 M PBS, 1 h 37°C.

11.PBS rinse, 3 .

12.Coverslip with Gelmount (Biomeda) or Aquamount (Gurr) as described above.

1.Human Retinal Sections

We have found that the microwave heat treatment can be too harsh (as detailed for GAPDH) and have often substituted a brief (about 10 min, room temperature) Neuropore permeabilization step instead or no permeabilization step depending on the quality of the case samples. Care trust be taken not to overexpose retinal sections to this agent.

We also find that there is a high level of nonspecific, dull background autofluorescence in human adult (aged) retina largely in the red range that we do not see in the brain/spinal cord sections. This is distinct from the bright autofluorescent granules that accumulate in retinal ganglion cell neurons. For this reason, we do not usually do more than one antibody on human postmortem sections

labeling for brightfield viewing. Unlike the Autoprobe original protocol, we have extended some of the times because of antigen accessibility in sections from old formalin-fixed blocks (as opposed to freshly perfused tissue). We have also increased the primary antibody incubation to overnight at 4°C. Note that the pepsin reagent digest times would likely need some changes in incubation times if used for human retinal sections.

1.Xylene 1—15 min.

2.Xylene 2—10 min.

3.100% Ethanol—10 min.

4.100% Ethanol—10 min.

5.Endoblocker (1 mL in 5 mL 100% ethanol), 5 min, room temperature.

6.95% Ethanol—3 min.

7.50% Ethanol—3 min.

8.d H2O—5 min.

9.Pepsin reagent 12 min at 37°C (prewarmed).

10.1 AutoBuffer (AB), three quick rinses.

11.Antigen Unmasking Solution (Vector labs) 3.77 mL stock AUS in 400 mL dist. H2O. Bring solution to boiling (in glass Pyrex measuring cup (4-cup size), at high power, about 5 min. Add 8–10 slides in plastic rack to solution, bring to boil in approximately 30 s (power setting 8) and then let boil for 1 min only. Let slides sit in buffer on the benchtop for 30–40 min.

12.1 AB 5 min RT

13.Tissue conditioner (Biomeda), 1 drop/mL AB, 10 min, room temperature.

14.Primary antibody—Caspase 3 (1 : 500, Pharmingen) or Bax (Santa

Cruz) (1 : 200) in primary antibody diluting buffer (Biomeda) O/N 4°C.

15.AB rinse, 2 5 min.

16.Secondary antibody, universal antibody (Biomeda kit), 40 min, 37°C

17.AB rinse, 2 .

18.Peroxidase reagent, 37°C, 30 min.

19.AB rinse, 1 .

20.Working chromogen solution, 15 min.

21.Dist H2O, stop bath, 2 5 min.

22.Coverlip with Gelmount or Aquamount (Gurr).

Useful information. You can likely find a different antigen retrieval method for every day of the month when you search the literature. We found that it helps to start with the mildest approach possible (i.e., use an unmasking solution at room temperature first) and then gear up to more aggressive heat treatments and then possibly choose to combine heat treatment with some type of enzymatic digest. We have found that we will vary our retrieval protocols according to

whether we are working with adult tissue or embryonic tissue (gentler methods), brain or retina (gentler methods). But most important is to find out what your antigen of interest does in the cell. Is it found in a membrane, is it bound to RNA, is it found in vesicles, in the nucleus? All of these will help determine what you need to do to make it accessible, often with a limited number of trial runs.

VI. METHODS FOR EXAMINING APOPTOTIC

CHANGES IN CULTURED CELLS

Many of our detection methods were first developed for in vitro models of apoptosis. These include ISEL/YOYO, measurement of mitochondrial membrane potential, immunocytochemistry for “apoptotic” proteins, DNA-binding dyes to visualize chromatin condensation, and so on. We generally use YOYO-1 for looking at chromatin condensation for tissue sections or cultured cells. This choice has really been determined by the capabilities of our krypton-argon laser. The Hoechst dye (bisbenziamide) and DAPI are also both excellent dyes for examining nuclear DNA and will not stain cytoplasmic RNA. Both are bright blue and require UV excitation, are long-lasting and slow to quench (see molecular probes). Either can be applied to tissue sections following antibody incubations in place of YOYO-1.

A.Hoechst Dye or YOYO-1 for Chromatin Condensation

For monolayer cell culture:

1.Wash cells 3 with PBS to remove serum/media.

2.Fix cells with 4% paraformaldehyde, 30 min on ice.

3.Rinse well with PBS, 3 .

4.Add Hoechst dye (5 g/mL PBS), 20–30 min at room temperature, in the dark. Or use YOYO-1 (1 : 1000 in PBS) for 20–30 min at room temperature, in the dark.

5.Wash 5 PBS.

6.Mount in Aquamount (Gurr). We have found that Gelmount is not a good choice for cultured cells stained with YOYO-1. It appears to “bleed out” of the cells after 24 h; therefore, we recommend Aquamount for mounting cell culture coverslips.

B.ISEL/YOYO for Monolayer Cell Cultures

1.Wash cells 3 with PBS to remove serum/media.

2.Fix cells with 4% paraformaldehyde, 30 min, on ice. With neuronal cultures we find it best to keep cells in fixative in the fridge overnight after the initial 30 min on ice, then rinse with PBS the next day.

3.Rinse well with PBS, 3 .

4.Dip in 100% methanol, 1–2 min, room temperature. It is important to keep this step short as apoptotic nuclei can dissociate from the remaining cell soma in cultured cells.

5.PBS rinse, 1 , 5 min.

6.RNAseA digest (100 µ/mL in 2 SSC), 10 min, 37°C

7.Rinse, 2 SSC, 2 5 min.

8.0.1 M glycine/0.1 M Tris, pH 7.2, 20 min RT. Note that the proteinase K digest is omitted (we found that it really served to detach cells from the coverslip rather than improve ISEL signal).

9.Equilibration buffer (Intergen), 15–30 min, room temperature.

10.TdT/BODIPY dUTP-red/reaction mix, 60 min, 37°C.

11.Rinse, 2 SSC (prewarmed), 3 10 min, 37°C.

12.PBS rinse, 2 5 min.

13.YOYO-1 (1 : 1000 in PBS), 20–30 min, room temperature, in the dark.

14.PBS rinses, 5 .

15.Aquamount coverslips, let dry in fume hood, then store in fridge.

We grow cells on 22-mm square coverslips for ISEL/YOYO or 12 mm round coverslips. The 22-mm coverslips are kept in 35-mm petri dishes and are easy to flush with different solutions. A parafilm coverslip can easily be applied to these in order to use a minimal volume of TdT reaction mix (30–40 µL). If using the round coverslip, we have found it easiest to transfer them to a ceramic well plate rather than keep them in a 24-well plastic tray. We invert them in each well, adding reagents under the coverslip (it will save on TdT reaction mix volume—use about 70 µL).

VII. MITOCHONDRIAL MEMBRANE POTENTIAL AS A MEASURE OF APOPTOTIC CHANGE

There is an increasing body of evidence that mitochondria play a critical decisional role in number of forms of apoptosis including those initiated in neurons by withdrawal of trophic factors, hypoxia/ischemia, and glutamate excitotoxicity. Using partially differentiated PC 12 cells, apoptosis is induced by removal of NGF and serum. We have found that there is a significant decline in mitochondrial membrane potential after trophic withdrawal, and that this precedes the appearance of nuclear apoptotic degradation.

We have used a fixable mitochondrial potentiometric dye, chloromethyltetramethylrosamine (CMTMR or Mitotracker orange) to measure Ψ∆M. Recently, the use of CMTMR and related dyes has been questioned (see Ref. 24). The mitochondrial uptake of cationic dyes used to estimate ψ∆M depends on the difference between the plasma membrane potential and Ψ∆M, and on the

time of incubation [25]. We [26–30] and others [31–34] have used rhodamine derivatives, with a chloromethyl moiety that binds to matrix thiols for this purpose. These derivatives offer the advantage that fixatives bind them to matrix proteins allowing immunocytochemistry to be subsequently carried out in the same cells. The purported disadvantage in using the fixable dyes has been thought to result from the dyes not being Nernstian in their outward movement from mitochondria (i.e., if Ψ∆M decreases during the incubation time, the dyes will not leave mitochondria in proportion to the extent of the decreases). Accordingly, the fixable dyes were proposed to offer less sensitive measures of Ψ∆M [24]. Furthermore, the binding of the fixable dyes to thiols has led to the consideration of the possibility that reactive oxygen species (ROS) levels rather than Ψ∆M might determine their in situ fluorescence [35] and that the dyes might damage mitochondria and open the PTP, dissipating Ψ∆M [36,37]. We have compared the sensitivity of the fixable dye CMTMR to other well-established mitochondrial potentiometric dyes and found that if cells are incubated with CMTMR for short periods before fixation (i.e., l5–30 min), they offer sensitive measures of Ψ∆M that are in agreement with those provided by other dyes such as JC-1. Ideally, Ψ∆M should be measured using more than one dye in order to confirm that observed changes in fluorescence can be reproduced. However other potentiometric dyes such as TMRM and JC-1 (molecular probes) require the capability of live cell imaging which may not be possible for many laboratories. For this reason we have only supplied a protocol for CMTMR.

CMTMR (Mitotracker Orange) enters mitochondria proportionally to the potential difference between the cytoplasmic compartment and mitochondrial matrix. After entering mitochondria, the chloromethyl groups of CMTMR react with thiols on proteins and peptides to form aldehyde-fixable conjugates and remain sequestered in the mitochondria after permeabilization and fixation [33,38]. The CMTMR mitochondrial fluorescence represents the highest level of potential difference between the mitochondrion and adjacent cytoplasmic compartment (or the nuclear compartment) of the living cell during the period of dye exposure prior to fixation. CMTMR fluorescence intensity as an estimate of Ψ∆M is measured and plotted as a frequency distribution (see Ref. 30 for details).

A.Mitotracker (Orange) plus YOYO-1

•20 50 µg vials; MW 427.37

•Add 850 µL sterile DMSO (N.B. — the full 850 µL will not fit in vial from Molecular Probes); gives a stock solution of 0.1 mM.

•Store as 25 µL aliquots at 20°C, protect from light and moisture.

1.Dilute mitotracker stock 1:1000 in prewarmed media.

2.Aspirate media from cells and add mitotracker-media for 15 min, return cells to 37°C, CO2 incubator.

3.Rinse 3 in HBSS.

4.Fix cells with 4% paraformaldehyde, for 20–30 min on ice.

5.Rinse 2–3 in PBS.

6.Expose cells for 1–2 min to methanol, room temperature.

7.Rinse cells with PBS, 2 .

8.Incubate cells with YOYO-1 (1:1000 in PBS) for 30 min, room temperature.

9.Rinse cells in PBS, 5 .

10.Mount coverslips with Aquamount. Once dry, store slides at 4°C.

B. Mitotracker, Bax (or GAPDH), and YOYO-1

Note that for cells that have been incubated with CMTMR and fixed, no detergent should be used in later steps (such as Triton or Tween) because it will result in CMTMR bleeding out of the mitochondria. CMTMR-treated cells may be stored in PBS at 4°C for at least 1 week, if necessary. In the following protocol we have used Bax antibody (Santa Cruz, N-20 fragment), Bcl-2 (Santa Cruz), or GAPDH (Chemicon), but virtually any antibody may be used that does not require a detergent permeabilization to access its antigen. Note that we have also exposed CMTMR-treated cells to a cold ethanol postfix without any problems, and we have also used a diluted (1:3) solution of Neuropore for 5 min when a permeabilization step was required with no damage to CMTMR.

1.Rinse cell-coverslips with PBS, 1 5 min.

2.Expose briefly to methanol, 1–2 min, room temperature.

3.Rinse in PBS, 2 5 min.

4.Block with 10% NGS/PBS for 20 min, room temperature.

5.Primary antibody Bax or Bcl-2 (1 : 200) or GAPDH (1 : 500) in 1% NGS/PBS, overnight, 4°C.

6.Rinse cells with PBS, 2 5 min.

7.Secondary antibody, goat anti-mouse or goat anti-rabbit Cy5-IgG (or

Alexa 690), 1 : 200 in 1% NGS/PBS for 1 h, room temperature (or 40 min, 37°C).

8.Rinse cells with PBS, 2 5 min.

9.YOYO-1 (1 : 1000 in PBS) for 20–30 min, room temperature.

10.Rinse cells with PBS, 5 .

11.Mount coverslips with Aquamount, let air dry, and then store at 4°C, in the dark.

This protocol allows for triple labeling of the cell. Note that to visualize Cy5, laser excitation of the fluorophore will be necessary. CMTMR emits in a range similar to Texas Red or TRITC; therefore, appropriate secondary antibody conjugates must be chosen that do not overlap with CMTMR or with YOYO-1. Alter-

nately, a double-label may be employed—CMTMR with YOYO-1 or DAPI, CMTMR with any other antibody using an Alexa 488 -IgG conjugate (if no confocal is available). Note that GAPDH is used at 1 : 500 dilution on cultured cells, but at 1 : 200 on tissue sections.

REFERENCES

1.Kerr JFR, Wyllie AH, Currie AR. Apoptosis, a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26:239–257.

2.Zou H, Henzel WJ, Liu XS, Lutschg A, Wang XD. Apaf-1, a human protein homologous to C-elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997; 90:405–413.

3.Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91:479–489.

4.Bratton SB, MacFarlane M, Cain K, Cohen GM. Protein complexes activate distinct caspase cascades in death receptor and stress-induced apoptosis. Exp Cell Res 2000; 256:27–33.

5.Liu XS, Zhou H, Slaughter C, Wang XD. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 1997; 89:175–184.

6.Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y, Tsujimoto Y. Acinus is a caspase 3-activated protein required for apoptotic chromatin condensation. Nature 1999; 401:168–173.

7.Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Constantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397:441–446.

8.Susin SA, Lorenzo HK, Zamzami N, Marzo I, Brenner C, Larochette N, Prevost MC, Alzari PM, Kroemer G. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J Exp Med 1999; 189:381–394.

9.Tatton NA, Maclean-Fraser A, Tatton WG, Perl DP, Olanow CW. A flourescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson’s disease. Ann Neurol 1998; 44: S142–148.

10.Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in-situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493–501.

11.Walker PR, Sikorska M. Eudonuclease activities, chromatin structure, and DNA degradationn in apoptosis. Biochem Cell Biol 1994; 72:615–623.

12.Cohen GM, Sun XM, Snowden RT, Dinsdale D, Skilleter DN. Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J 1992; 286:331–334.

13.Oberhammer F, Wilson JW, Dive C, Morris ID, Hickman JA, Wakeling AE, Walker PR, Sikorska M. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/

or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO 1993; 12:3679–3684.

14.Peitsch MC, Polzar B, Stephan H, et al. Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis programmed cell death. EMBO 1993; 12:371–377.

15.Deibel M, Coleman MS. Biochemical properties of purified human terminal deoxynucleotidyltransferase. J Biol Chem 1980; 255:4206–4212.

16.Tabor S, Struhl K, Scharf SJ, Gelfand DH. Enzymatic manipulation of DNA and RNA. In: Current Protocols in Molecular Biology. New York: Wiley & Sons, Inc, 1999.

17.Didenko VV, Hornsby PJ. Presence of double-strand breaks with single-base 3′overhangs in cells undergoing apoptosis but not necrosis. J Cell Biol 1996; 135:1369– 1376.

18.Dypbukt JM, Ankarcrona M, Burkitt M, Sjoholm A, Orrenius S, Nicoteri P. Different pro-oxidant levels stimulate growth, trigger apoptosis or produce necrosis of insulin-secreting RINmSF cells: the role of intracellular polyamines. J Biol Chem 1994; 269:30553–30560.

19.Funk A, Rudel J, Fellbaum C, Hofler H. Specific in situ end labeling of apoptosis shows different rates of programmed cell death in non-Hodgkin lymphomas. Verh Dtsch Ges Pathol 1994; 78:318–320.

20.Tatton NA. Increased caspase 3 and bax immunoreactivity accompany nuclear GAPDH translocation neuronal apoptosis in Parkinson’s Disease. Exp Neurol 2000; 166:29–43.

21.Tatton NA, Kish SJ. In situ detection of apoptotic nuclei in the substantia nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using terminal deoxynucleotidyl transferase labelling and acridine orange staining. Neuroscience 1997; 77:1037–1048.

22.Ishitani R, Chuang DM. Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons. Proc Natl Acad Sci USA 1996; 93:9937–9941.

23.Carlile GW, Chalmers-Redman RM, Tatton NA, Pong A, Borden KE, Tatton WG. Reduced apoptosis after nerve growth factor and serum withdrawal: conversion of tetrameric glyceraldehyde-3-phosphate dehydrogenase to a dimer. Mol Pharmacol 2000; 57:2–12.

24.Bernardi P, Scorrano L, Colonna R, Petronilli V, Di Lisa F. Mitochondria and cell death: mechanistic aspects and methodological issues. Eur J Biochem 1999; 264: 687–701.

25.Rottenberg H, Wu S. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim Biophys Acta 1998; 1404:393–404.

26.Chalmers-Redman RM, Fraser AD, Carlile GW, Pong A, Tatton WG. Glucose protection from MPP -induced apoptosis depends on mitochondrial membrane potential and ATP synthase. Biochem Biophys Res Commun 1999; 257:440–447.

27.Grant SM, Shankar SL, Chalmers-Redman RM, Tatton WG, Szyf M, Cuello AC. Mitochondrial abnormalities in neuroectodermal cells stably expressing human amyloid precursor (hAPP751). Neuroreport 1999; 10:41–46.

28.Jing Y, Dai J, Chalmers-Redman RM, Waxman S. Arsenic troxide selectively in-