Zinc Fixation for Flow Cytometry UNIT 7.

40
Analysis of Intracellular and Surface
Epitopes, DNA Content, and Cell
Proliferation
Rikke Christensen,1 David M. Owens,2 Anette Thomsen,3 Søren Pedersen,1 and
Uffe Birk Jensen1,3
1
Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark
2
Departments of Dermatology and Pathology, College of Physicians and Surgeons, Columbia
University, New York, New York
3
Department of Human Genetics, Aarhus University, Aarhus, Denmark

ABSTRACT
Zinc salt-based fixation (ZBF) is a simple, cost-effective, and nonhazardous fixation
method for cell suspensions that preserves all cellular structures and enables flow cy-
tometric analysis of both surface and intracellular proteins, DNA content profiles, and
pulse-labeling using the thymidine analog EdU in the same cell sample. ZBF performs
equally well to formaldehyde in the preservation of surface epitope labeling and forward
and side light scatter parameters, as measured by flow cytometry. DNA is maintained at
high molecular weight, improving the quantification and allowing subsequent quantita-
tive PCR analysis. Finally, ZBF treatment allows for long-term storage of labeled cells
with little change in these parameters. Curr. Protoc. Cytom. 57:7.40.1-7.40.9. 
C 2011 by

John Wiley & Sons, Inc.

Keywords: DNA content r DNA synthesis r Click-iT assay r
surface and intracellular markers

Zinc salt-based fixation (ZBF) is a simple and nonhazardous method that can replace BASIC
formaldehyde-based fixation in many applications. ZBF is well suited for fixation of PROTOCOL
single cells for flow cytometry. Cross-linking agents, such as normal buffered formalin
(NBF) and paraformaldehyde (PFA), are known to cause DNA fragmentation and changes
in the tertiary and quaternary structure of proteins (Mason and O’Leary, 1991; Gillio-Tos
et al., 2007). These obstacles are reduced with the use of ZBF as zinc salts preserve RNA,
DNA, and proteins to a higher extent than other fixatives (Wester et al., 2003; Lykidis
et al., 2007). Furthermore, ZBF has been shown to result in improved immunolabeling
of sensitive epitopes.

In many applications, it is useful to be able to combine analysis of surface and intra-

cellular markers with measurements of cell proliferation and DNA content. Classically,
measurement of cell proliferation has been performed via BrdU-labeling. However, BrdU
detection requires HCl or DNase treatment in order to generate single-stranded DNA that
can be accessed by BrdU antibodies. This process can be incompatible with maintenance
of intact DNA profiles. In addition, many antigen epitopes are sensitive to HCl treatment
(Tang et al., 2007). With the introduction of the new thymidine analog, EdU, and “click
chemistry,” it is now possible to study cell proliferation without using agents that de-
nature or digest the DNA (Cappella et al., 2008), and the Click-iT protocol works well
on zinc-fixed cells. Thus, ZBF enables flow cytometric analysis of both cell surface and
intracellular proteins, DNA content profiles, and DNA replication using EdU in the same
cell sample. Nucleic Acid
Analysis

Current Protocols in Cytometry 7.40.1-7.40.9, July 2011 7.40.1

Published online July 2011 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/0471142956.cy0740s57 Supplement 57
Copyright C 2011 John Wiley & Sons, Inc.
 One major advantage using this method is the long-term cell stability at −20◦ C. This
enables collection of cell samples at different time points over long periods of time and
analysis of all collected samples at the same time. Thereby, errors such as day-to-day
variation in cell staining and data collection can be avoided. No upper limit in storage
duration has yet been observed when the cells are stored at −20◦ C.

Cells fixed using ZBF can be stained using different protocols that can be combined
according to need. Here, we describe a protocol that combines the Click-iT proliferation
assay with cell cycle analysis and antibody stainings on zinc-fixed cells. Each of the
sections in this protocol can also be applied individually. This protocol describes a
nonhazardous fixation method that can be used on single-cell suspensions isolated from
blood or tissue, as well as cell cultures.

Materials
10 mM EdU (5-ethynyl-2 -deoxyuridine; Invitrogen, cat. no. A10044) dissolved in
DMSO; store up to 1 year at −20◦ C
Primary cells isolated from fresh tissue or cells cultured in appropriate cell culture
medium
10 mg/ml EdU dissolved in 0.9% NaCl (prepare fresh)
Phosphate-buffered saline, without calcium and magnesium (CMF-PBS), pH 7.2
Zinc salt-based fixation buffer (ZBFB; see recipe)
Glycerol
Tris-buffered saline (TBS), pH 7.4
Permeabilization buffer (see recipe)
Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay kit (Invitrogen, cat.
no. 35002)
Antibodies:
PC7-conjugated CD4 (Beckman Coulter, cat. no. 737660)
PE-conjugated CD8 (DAKO, cat. no. R0806)
FITC-conjugated CD14 (DAKO, cat. no. F0844)
APC-conjugated CD45 (DAKO, cat. no. C7230)
PE-Cy7-conjugated Ly-6A/E (Sca-1; BD Pharmingen, cat. no. 558162)
AlexaFluor 647-conjugated CD34 (eBiosciences, cat. no. 51-0341-82)
Mouse monoclonal antikeratin 10 antibody (LHP2, a generous gift from I. Leigh)
AlexaFluor 700 goat anti–mouse IgG (H+L) (Molecular Probes, cat.
no. A21036)
Wash buffer (see recipe)
Ice
Hoechst staining solution (see recipe)
37◦ C CO2 incubator
0.5-ml Myjector U-100 insulin syringes, 27-G needle (BD Biosciences)
Centrifuge
Vortex
4◦ C incubator
2-ml microcentrifuge tubes
Rocking table
Flow cytometer equipped with three lasers: Near-UV 375 nm, blue 488 nm, and red
633 nm
NOTE: All protocols using live animals must first be reviewed and approved by an Insti-
tutional Animal Care and Use Committee (IACUC) and must follow officially approved
procedures for the care and use of laboratory animals.
Zinc Fixation for
Flow Cytometry

7.40.2
Supplement 57 Current Protocols in Cytometry
Prepare the cells for ZBF fixation
1a. For cell cultures: Add 10 mM EdU stock solution (diluted in DMSO to a final
concentration of 10 μM) to the cell culture medium and incubate the cells for 30 to
60 min at 37◦ C in a CO2 incubator. Harvest the cells using a standard trypsinization
protocol to obtain a single-cell suspension.
Cell lines that have long doubling times may require longer EdU exposure.

1b. For in vivo injection in mice: Using a 0.5-ml Myjector syringe equipped with a 27-G
needle, inject 2 mg EdU of a 10 mg/ml stock solution diluted in 0.9% NaCl s.c.
into mice that weigh ∼20 g. Sacrifice the mice by cervical dislocation 2 hr later and
isolate the desired cell type.
When shorter or longer exposure times with EdU are required, the amount of EdU
administered can be increased or reduced.
2. Collect the cells in a centrifuge tube, pellet by centrifuging 5 min at 200 × g, 4◦ C,
and remove the supernatant.
If cell proliferation analysis is not desirable, treat and harvest the cells as usual and omit
step 1.

3. Resuspend the cells at 5–10 × 106 cells in 100 μl CMF-PBS and mix well.
For optimal fixation, it is important to avoid clumps.

Fix the cells with ZBF

4. Vortex the tube containing the dislodged cell pellet for 5 sec and add 1 ml ZBFB
slowly during continued vortexing. Vortex for an additional 5 sec after adding the
ZBFB.
Vortexing at medium speed is important to avoid clumping of the fixed cells.

5. Incubate the cells overnight at 4◦ C.

If preservation of RNA is preferred, the ZBF-fixed cells should be directly frozen in glycerol
in a 1:1 ratio after addition of ZBFB and stored immediately at −20◦ C.
Store the fixed cells for long-term (optional)
6. Add glycerol in a 1:1 ratio and mix carefully.
7. Store the samples at −20◦ C.
The samples can be stored for at least a year without loss of epitopes. Since the sample
does not freeze due to the glycerol, aliquots can be taken out at different time points
without thawing. It is important to mix or vortex the tube before removing the aliquot.
The cells can also be stored for 2 to 3 weeks at 4◦ C in the ZBFB, without addition of
glycerol.

Remove the ZBFB buffer

8. Transfer the desired amount of cells needed for downstream applications into a 2-ml
microcentrifuge tube.
9. Add 1 ml TBS and mix well. Centrifuge 3 min at 1000 × g, room temperature.
10. Discard the supernatant, dislodge the cell pellet by flicking the tube, and add 1 ml
TBS.
11. Place the tube on a rocking table with gentle rocking for 15 to 30 min.
12. Centrifuge 3 min at 1000 × g, room temperature, and repeat steps 10 and 11 once.
13. Divide the cells into the desired amount of tubes. Nucleic Acid
Analysis
Each tube should contain ∼1 × 106 cells.
7.40.3
Current Protocols in Cytometry Supplement 57
 14. Centrifuge 3 min at 1000 × g, room temperature, and discard the supernatant.
If only antibody stainings for surface markers are performed on the cells, permeabilization
may be omitted (proceed to step 24). However, depending on the cell type and condition of
the cells before fixing, the cells may be partly permeable, which can lead to trapping of the
antibodies inside the cells. If unexpected staining patterns are observed, we recommend
the permeabilization steps.

Permeabilize the cells

15. Dislodge the cell pellet and resuspend the cells in 500 μl permeabilization buffer.
We use saponin to permeabilize the cells since permeabilization agents, such as Triton-X,
may result in complete loss of certain surface epitopes.

16. Incubate for 30 min at room temperature, protected from light.

Since EdU is light sensitive, protect from light when using EdU, otherwise protection from
light is not necessary.

17. Centrifuge 3 min at 1000 × g, room temperature, remove the supernatant, and
dislodge the pellet.
If antibody stainings for intracellular markers are performed without EdU detection,
proceed to step 24.

Perform Click-iT staining

18. Prepare the Click-iT reaction cocktail according to the instructions in the kit.
The cocktail should be used within 15 min of preparation.
19. Add 0.5 ml Click-iT reaction cocktail to each tube and mix well.
20. Incubate for 30 min at room temperature in the dark.
21. Add 1 ml permeabilization buffer and mix well.
22. Centrifuge 3 min at 1000 × g, room temperature, and discard the supernatant.
23. Repeat steps 21 and 22 two additional times.
IMPORTANT NOTE: The Cu ions from the Click-iT reaction cocktail may influence some
fluorochromes. Therefore, careful washing at this step is crucial for downstream antibody
stainings.

Perform antibody staining

24. Prepare the antibody staining solution, e.g., PE-Cy7-conjugated Ly-6A/E (1:100),
AlexaFluor 647-conjugated CD34 (1:50), and a mouse monoclonal anti-keratin
10 antibody (1:100, LHP2) in 100 μl permeabilization buffer.
25. Add 0.1 ml antibody solution to the each tube and leave for 30 min on ice in the
dark.
Incubation times and concentrations for primary and secondary antibodies should be
optimized for individual antibodies.

26. Wash the cells three times, each time with 500 μl wash buffer and centrifuge 3 min
at 1000 × g, room temperature.
27. Resuspend the cell pellet in 100 μl of secondary antibody (e.g., AlexaFluor
700-conjugated secondary antibody) diluted in permeabilization buffer.
Goat anti–mouse antibody diluted 1:400 was used in this example.

28. Incubate for 30 min on ice in the dark.

Zinc Fixation for
Flow Cytometry 29. Wash the cells two times, each time with 500 μl wash buffer and centrifuge 3 min at
1000 × g, room temperature.
7.40.4
Supplement 57 Current Protocols in Cytometry
 If cell cycle analysis is not foreseen, cells can be resuspended in 500 μl permeabilization
buffer and subjected to flow cytometry analysis. Otherwise, continue with step 30.
Perform cell cycle analysis
30. Resuspend the cell pellet in 500 μl Hoechst staining solution.
31. Incubate for 15 to 60 min at room temperature, protected from light.
Incubation times and the concentration of Hoechst should be optimized for each cell
type. To shorten the procedure, Hoechst can also be added in step 24 together with the
antibodies. After washing off excess antibodies, Hoechst should be added again to the
cell suspension.
Perform flow cytometric analysis
32. Using 375-nm excitation, collect the emitted light from Hoechst through a
424/42 filter (or similar). Alexa 488, PE, and PE-Cy7 are excited by a 488-nm
laser. Collect the emitted light through 530/30, 585/42, and 780/60 band-pass filters
(or similar), respectively. Alexa 647 and Alexa 700 are excited by the 633-nm laser.
Collect the emitted light through 660/20 and 730/45 band-pass filters (or similar),
respectively.
For cell cycle studies, doublet discrimination can be achieved by pulse processing. Create
a dot plot of pulse area against pulse width. A doublet of cells in G1 will have the same
pulse area as a single cell in G2/M but the pulse width will be much greater, allowing
appropriate gating on the dot plot.
Data on DNA content/cell cycle is detected with linear amplification whereas the fluores-
cent signals from the antibody stainings are detected with logarithmic amplification.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Hoechst staining solution

Hanks buffered salt solution (HBSS) containing:
1 μM Hoechst 33342
0.1% (w/v) BSA
0.2% (w/v) saponin
Prepare fresh
Permeabilization buffer
Phosphate-buffered saline (PBS), pH 7.2, containing:
0.1% (w/v) BSA
0.2% (w/v) saponin
Store up to 2 months at 4◦ C
Wash buffer
Phosphate-buffered saline (PBS), pH 7.2, containing:
0.2% (w/v) saponin
Store up to 2 months at 4◦ C
Zinc-based fixation buffer (ZBFB)
0.1 M Tris·Cl, pH 7.8 (APPENDIX 2A)
0.05% (v/v) calcium acetate (CH3 COO)2 Ca
0.5% (v/v) zinc acetate (CH3 COO)2 Zn
0.5% (v/v) zinc chloride ZnCl2 Nucleic Acid
Analysis
Store up to 1 year at room temperature
7.40.5
Current Protocols in Cytometry Supplement 57
 COMMENTARY
Background Information
The main purpose of chemical fixation is
to preserve tissue and subcellular architec-
ture, thereby facilitating immunohistochemi-
cal detection of epitopes and histochemical de-
tection of chemical constituents within cells.
Formaldehyde-based fixatives fulfill this re-
quirement to a large extent and have been the
preferred compounds for decades in standard
pathology procedures. However, aldehyde-
based fixatives often result in poor antigen
preservation due to their cross-linking mecha-
nism and DNA fragmentation (Cattoretti et al.,
1993; Gillio-Tos et al., 2007). Furthermore,
aldehyde-based fixatives can interfere with
DNA dye binding resulting in DNA content
histograms of poor quality (Rousselle et al.,
1998). In histopathology, there has been in-
creased focus on using non-aldehyde-based
fixatives (zinc salt fixatives) because they com-
bine good morphological preservation with
good antigen preservation (Beckstead, 1994;
Wester et al., 2003; Hicks et al., 2006; Lykidis
et al., 2007). In addition, DNA and RNA
are maintained intact to a higher extent us-
ing zinc-based fixatives than aldehyde-based
fixatives (Lykidis et al., 2007; Jensen et al.,
2010). The molecular mechanism behind ZBF
is not yet well understood. However, zinc
salts have been used for decades as addi-
tives in formaldehyde-based fixatives in order
to improve the morphology, and it is known
that Zn ions can stabilize certain parts of the
A B
105 105
K10 AlexaFluor700-A
104 104
CD34 APC-A
103 103
2 2
10 10
0 0
0 102 103 104 105 0 102
Sca-1 PE-Cy7-A
Figure 7.40.1 Analysis of surface (A) and intracellular (B) antibody-stained pulse-labeled cells and DNA profiles
(C), on the same zinc-fixed sample. EdU (2 mg/ml) was administered s.c. and the mouse sacrificed 2 hr later. Anti-
bodies against Sca-1 and CD34 were used to stain the surface markers and an antibody against keratin 10 was used to
stain an intracellular marker. Hoechst was used to stain the DNA.
Zinc Fixation for
Flow Cytometry
7.40.6
Supplement 57
tertiary structure of proteins when subjected
to chaotropic agents, such as acetate (Selevsek
et al., 2009). In the present unit, we describe a
method for fixation of single-cell suspensions
using zinc salts. The advantage of this method
is that multiple parameters can be analyzed on
the same cell sample because proteins, DNA,
and RNA are preserved.
The zinc fix method can be combined with
the Click-iT EdU AlexaFluor 488 Flow Cy-
tometry Assay, as described in the Basic Pro-
tocol. One of the drawbacks of using the kit
is that cells are fixed with paraformaldehyde
and 7-AAD is used as a DNA-binding dye.
These steps cause loss of resolution and broad-
ening of the G0/G1 peak in the DNA con-
tent histograms (Rousselle et al., 1998). When
zinc salts are used for fixation, the DNA is
preserved to a higher degree, as problems
with cross-linking and DNA fragmentation are
avoided.
Using aldehyde-based fixatives, samples
can normally be stored up to 1 week at
4◦ C. A major advantage of the zinc fixa-
tion method is that samples can be stored
for more than 1 year at −20◦ C (the sample
analyzed in Fig. 7.40.1 has been stored for
nearly 2 years at −20◦ C). This enables collec-
tion of cell samples at different time points
over long periods of time. Analysis of the
samples can subsequently be performed at the
same time avoiding errors such as day-to-day
variation.
C
105
104
EdU FITC-A
103
35.6
102
0
103 104 105 0 1000 2000 3000 4000
Sca-1 PE-Cy7-A DNA Hoechst Blue-A
Current Protocols in Cytometry
Critical Parameters and
Troubleshooting
To avoid clumping when adding the ZBFB
buffer, it is very important that cells be dis-
lodged and mixed properly by vortexing prior
to fixation. Zinc-fixed clumps are difficult to
redissolve. Clumping may result in uneven cell
staining and cell loss. Eventual cell clumps
should be removed by filtering before analysis.
It is important to wash out glycerol and zinc
properly before proceeding with the stainings
to avoid interference of zinc with the subse-
quent analysis, as the added antibodies will
loose their properties if remnants of the fix-
ative are present. When the cells have been
frozen in glycerol, it may be more difficult to
sediment the cells in the first wash. Make sure
that the cell pellet is of the expected size after
the first centrifugation. Repeat the centrifuga-
tion with a higher g force if the pellet appears
too small.
Some fluorochromes are sensitive to the
Click-iT reaction. If the separation between
stained and unstained cells is too low, it is

A B C
4000 4000 4000

3000 3000 3000

SSC-A

SSC-A

SSC-A
2000 2000 2000
6.66 3.19 9.44
1000 1000 1000

15.3 26.7 32.5

0 0 0
0 1000 2000 3000 4000 0 1000 2000 3000 4000 0 1000 2000 3000 4000
FSC-A FSC-A FSC-A

105 105 105

104 104 104

<PE-A>: CD8

<PE-A>: CD8

<PE-A>: CD8
103 103 103

102 102 102

0 0 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105
<PE-Cy7-A>: CD4 <PE-Cy7-A>: CD4 <PE-Cy7>: CD4

105 105 105

<APC-A>: CD45

<APC-A>: CD45

<APC-A>: CD45

104 104 104

103 103 103

102 102 102

0 0 0

0 102 103 104 105 0 102 103 104 105 0 1000 2000 3000 4000
<FITC-A>: CD14 <FITC-A>: CD14 <FITC-A>: CD14

Figure 7.40.2 Comparison of scatter parameters and surface stainings in a live (A), PFA-fixed (B), or zinc-fixed (C) whole
blood sample. Scatter parameters are shown in the upper panel. In the middle panel, cells are stained with antibodies
against the surface markers CD4 and CD8 and in the lower panel cells are stained with antibodies against the surface
markers CD14 and CD45. Antibody stainings of gated populations are shown in blue (monocytes) and red (lymphocytes).
For the color version of this figure go to http://www.currentprotocols.com/protocol/cy0740.

7.40.7
Current Protocols in Cytometry Supplement 57
 recommended that cells remain for a few min-
utes in the permeabilization buffer during the
washing steps after the Click-iT reaction.
EdU has been reported to have a slightly
increased anti-proliferative activity when used
for prolonged exposures (Cappella et al.,
2008). For short exposures, such as 1 hr, the
growth inhibition is negligible. When using
EdU for in vivo applications, we have observed
that EdU is removed more rapidly from the cir-
culation than BrdU (Jensen et al., 2010).
Anticipated Results
Figure 7.40.2 shows a typical result of an
analysis of the surface markers CD4, CD8,
CD14, and CD45 in a whole blood sample that
has been fixed in either ZBFB (panel C) or PFA
(panel B). In panel A, the analysis has been
performed on live cells. The light scatter sig-
nals in the zinc-fixed cells are more similar to
the light scatter signals in live cells compared
to PFA-fixed cells. Most importantly, lympho-
cytes, monocytes, and granulocytes can still
be recognized as separate populations. The
minor differences may be explained by small
changes in size and granularity of the cells.
The staining pattern of the surface markers
in the zinc-fixed sample also resembled the
pattern in live cells more than the pattern in
PFA-fixed cells.
Since a major advantage of this method is
that multiple stainings can be performed on the
same sample, Figure 7.40.1 shows an exam-
ple in which cell proliferation, DNA content,
surface and intracellular markers are analyzed
in one sample of epidermal keratinocytes iso-
lated from EdU-labeled C57Bl/6 mice. Panel
A shows the flow cytometric analysis of the
surface markers Sca-1 and CD34 and panel
B analysis of the intracellular marker keratin
10. Finally, in panel C, incorporated EdU is
detected using the Click-It EdU Alexa488 kit
and Hoechst 33342 is used to stain the DNA.
In the example shown, the sample has been
stored in the freezer for 21 months. The pat-
tern is similar to the pattern in freshly fixed
cells.
Time Considerations
When the cells are ready for fixation, the
ZBF procedure only requires 1 min of hands-
on time. However, for proper fixation the cells
should be incubated overnight. Removal of
the ZBFB requires ∼1 hr. Permeabilization
requires ∼35 min. The Click-iT reaction re-
quires ∼45 min. Antibody stainings require
Zinc Fixation for
Flow Cytometry 45 min to 1.5 hr (depending on whether conju-
7.40.8
Supplement 57
gated antibodies are used or incubation with a
secondary antibody is necessary). DNA stain-
ing requires ∼30 min. The indicated times are
total times including incubation. The hands-on
time is significantly shorter.
Acknowledgement
We kindly acknowledge support from the
following: Aage Bang Fund, Karen Elise
Jensen Fund, and Lundbeck Fund.
Literature Cited
Beckstead, J.H. 1994. A simple technique for
preservation of fixation-sensitive antigens in
paraffin-embedded tissues. J. Histochem. Cy-
tochem. 42:1127-1134.
Cappella, P., Gasparri, F., Pulici, M., and Moll, J.
2008. A novel method based on click chemistry,
which overcomes limitations of cell cycle anal-
ysis by classical determination of BrdU incor-
poration, allowing multiplex antibody staining.
Cytometry A 73:626-636.
Cattoretti, G., Pileri, S., Parravicini, C., Becker,
M.H., Poggi, S., Bifulco, C., Key, G., D’Amato,
L., Sabattini, E., Feudale, E., Reynolds, F.,
Gerdes, J., and Rilke, F. 1993. Antigen unmask-
ing on formalin-fixed, paraffin-embedded tissue
sections. J. Pathol. 171:83-98.
Gillio-Tos, A., De Marco, L., Fiano, V., Garcia-
Bragado, F., Dikshit, R., Boffetta, P., and
Merletti, F. 2007. Efficient DNA extraction from
25-year-old paraffin-embedded tissues: Study of
365 samples. Pathology 39:345-348.
Hicks, D.J., Johnson, L., Mitchell, S.M., Gough, J.,
Cooley, W.A., La Ragione, R.M., Spencer, Y.I.,
and Wangoo, A. 2006. Evaluation of zinc salt
based fixatives for preserving antigenic determi-
nants for immunohistochemical demonstration
of murine immune system cell markers. Biotech.
Histochem. 81:23-30.
Jensen, U.B., Owens, D.M., Pedersen, S., and
Christensen, R. 2010. Zinc fixation preserves
flow cytometry scatter and fluorescence pa-
rameters and allows simultaneous analysis of
DNA content and synthesis, and intracellu-
lar and surface epitopes. Cytometry A 77:798-
804.
Lykidis, D., Van Noorden, S., Armstrong, A.,
Spencer-Dene, B., Li, J., Zhuang, Z., and Stamp,
G.W. 2007. Novel zinc-based fixative for high
quality DNA, RNA and protein analysis. Nucleic
Acids Res. 35:e85.
Mason, J.T. and O’Leary, T.J. 1991. Effects of
formaldehyde fixation on protein secondary
structure: A calorimetric and infrared spectro-
scopic investigation. J. Histochem. Cytochem.
39:225-229.
Rousselle, C., Robert-Nicoud, M., and Ronot, X.
1998. Flow cytometric analysis of DNA content
of living and fixed cells: A comparative study
using various fixatives. Histochem. J. 30:773-
781.
Current Protocols in Cytometry
Selevsek, N., Rival, S., Tholey, A., Heinzle, E.,
Heinz, U., Hemmingsen, L., and Adolph, H.W.
2009. Zinc ion-induced domain organization in
metallo-beta-lactamases: A flexible “zinc arm”
for rapid metal ion transfer? J. Biol. Chem.
284:16419-16431.
Tang, X., Falls, D.L., Li, X., Lane, T., and
Luskin, M.B. 2007. Antigen-retrieval procedure
for bromodeoxyuridine immunolabeling with
concurrent labeling of nuclear DNA an anti-
gens damaged by HCl pretreatment. J. Neurosci.
27:5837-5844.
Wester, K., Asplund, A., Backvall, H., Micke, P.,
Derveniece, A., Hartmane, I., Malmstrom, P.U.,
and Ponten, F. 2003. Zinc-based fixative im-
proves preservation of genomic DNA and pro-
teins in histoprocessing of human tissues. Lab.
Invest. 83:889-899.

Nucleic Acid
Analysis

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Current Protocols in Cytometry Supplement 57