Immunostaining of chromosomal proteins in plant cells
 
Chromosomal Proteins

 These protocols for immunolabelling (US: immunolabelling ) were used to test antibodies to mostly human chromosomal proteins such as CENP-E, acetyl-histone H3 and phospho-histone H3 in Arabidopsis plant cells and cultures, where they label histone H3 and other histones, phosho-histone H3, and acetylated histones. See the antimethylcytosine antibody labelling page for using that antibody to methylated DNA. The protocols for tubulin / microtubules are also given.

Futher details of chromosome preparation and in situ hybridization protocols are found in our book, Practical in situ Hybridization, 2000. T Schwarzacher and P Heslop-Harrison, published by BIOS and available from Sigma Chemical (for c. GBP30, USD$50, JPY10,500 or other currencies) or from any bookshop. This is the 'book' referred to below and familiarity/cross references to these methods is assumed. If there is another addition, this protocol will probably be included.

 

Buffers and solutions

2xMT buffer

10 ml      1 M PIPES pH6.8
10 ml      100 mM MgSO4
10 ml      100 mM EGTA

70 ml       H2O

8%PFA
8 g paraformaldehyde in 80 ml water, heat for 30 min at 60C, and add 2-3drops of 1 N NaOH or KOH to clear and  make to 100ml. Adjust pH to 7 with H2SO4 . Aliquot and freeze at -20C or keep at 4C for use in 1-2 days (see book Protocol 8.1)
10x KPBS

1.28M NaCl

20mM KCl

80mM Na2HPO4

20mM KH2PO4

pH 7.2

This is different to book appendix 1 (PBS)

Digestion
enzymes
As book, protocol 5.3
Taxol
100uM stock solution of Taxol (e.g. Sigma Paclitaxel) in DMSO (keep at 4C but warm to room temperature and agitate before use as it precipitates). Taxol stabilizes microtubules, and the DMSO both dissolves the taxol and helps penetration of all fixative components
Other solutions

:DAPI at 1ug/ul in KPBS as chromosome counter stain

Triton X-100

Tween-20

0.1% sodium borohydride in KPBS (freshly made immediately before use)

BSA block: 1% BSA Fraction 5, essentially IgG free, in KPBS optionally containing 01-0.2% Tween-20 (see book for discussion of BSA grades)

Antibody solution: 0.1% Acetylated BSA in KPBS/0.1-0.2% Tween-20 with appropriate antibody, typically 1:200 to 1:1000 dilution.

Poly-L-lysine coated slides; make your own (see book)  or buy ready coated from Sigma or other suppliers.

Fixation
 

For Microtubules: Fix young Arabidopsis inflorescences (groups of 5-10 buds) or cell suspension in MT fix (2ml MT buffer, 2ml PFA, 8ul Triton X-100, 8ul Taxol) for 1hour at plant growth temperature.

For Chromatin Proteins: Fix young inflorescences or cells in KPBS fix: 2ml PFA, 800 ul 10xKPBS, 1.2 ml water, 8 ul Triton X-100, 8ul Taxol (used here for the benefit of the DMSO)

Chromosome Preparation
 

The aim is to get chromosomes well spread and free of cytoplasm. Much advice is given in the book 'Practical in situ hybridization' chapter 5, although without acid fixation (which would destroy proteins) it is difficult to get good spreads. 

For Microtubules: Macerate material in 0.5% Triton X-100 with the ends of a glass or metal rod, place on poly-L-lysine coated slide and squash under a coverslip, aiming to get a monolayer of cells, still intact in their walls. Check metaphase index by counterstaining with DAPI after removing coverslip by freezing.  Check cell walls by phase-contrast microscopy. Alternatively, make a cell monolayer in water and let dry down onto poly-L-lysine coated slide.

For Chromatin Proteins: Dissect the material in a drop of 0.5% Triton X-100, and place tissue with metaphases onto a poly-L-lysine coated slide. Squash under coverslip, or macerate with a glass/metal rod before putting on coverslip. Freeze slide and flick off coverslip. To look for chromosomes/metaphases, add a drop of DAPI stain and examine under UV microscope.

It is probably best to not allow preparations to dry. After removing coverslip by freezing, place into cuvette of KPBS for up to 4 h. Mark area of preparation by scratching (see book).

Post-Fixation
 

Optionally, re-fix preparations in fresh paraformaldehyde (Protocol 8.1 in book, stage 6 paraformaldehyde fixation only) and wash in KPBS.

If autofluorescence is likely to be a problem (e.g. from cell walls or chloroplasts), place 250 ul of 0.1% sodium borohydride solution onto the maked area of each slide and cover with a plastic coverslip. Once it has stopped fizzing (2-5 min), wash slides well through 4 changes of KPBS.

Antibody incubation
 

Block preparation by placing 250 ul BSA block onto the marked area of each slide, cover with a plastic cover slip (see book) and leave at room temperature in a humid chamber for 30 min,

Shake off block and add primary antibody solution (50-100 ul per slide). Cover with plastic coverslip and place in humid chamber for 1 hr  at 37C or 16-40h at 4C - typically overnight at 4C.

Detection, Counterstaining and Mounting
 

Wash four times in KPBS (optionally with 0.1-0.2% Tween-20), then follow protocols as protocol 9.2 for detection using appropriate secondary antibodies. Use 1% BSA in KPBS wityh 0.1% Tween as block, and make up antibodies in 0.1% Acetylated BSA in KPBS/Tween.

Follow protocol 9.3 for countertaining and mounting.

www.le.ac.uk

 

Contacts: Trude Schwarzacher and Pat Heslop-Harrison
 

Homepage: http://www.molcyt.com, this page is under the 'methods' sidebar.
e-mails: TS32(a)le.ac.uk and PHH4(a)le.ac.uk


University of Leicester homepage: June 2005