C banding of chromosome - staining problem (Apr/30/2006 )

Hi all,
I have problem regarding C banding of human chromosomes. I have cultured Human blood and have bearutiful metaphases on slides.
I follow the protocol for C banding is
0.2 M HCl for 20-30 min
Rinse with DI water
4% Ba(OH)2 at 60 c for 8-15 sec
Rinse
2x SSC at 60 C for 20min
Rinse
1:5 diluted Leishmans stain (diluted with Gurr Buffer, commercila tablets) for 1min
Rinse

MY problem is, chromosome gets stained all over rather than staining only at centromere.
I tried diff timings in Ba(oH)2 and I always use strain, that is made fresh.
Can anyone please help? I am really stressed.
Many thanks
Vibha.

Vibha
I have cut and pasted our method below which is a close variation of yours. If you want to try your own method again perhaps bake the slide on a hotplate for 15 mins at 75C prior to C banding to see if this helps (assuming your slides are fresh). Otherwise your protocol appears OK. The only other suggestions are to use a fresh BaOH2 solution and extend time.
Good luck.


C BANDING

PRINCIPLE
C-banding stains constitutive heterochromatin which is present around the centromeres of all human chromosomes, and is most abundant around the centromeres of chromosomes 1, 9, 16 and the distal long arm of the Y chromosome.

C-banding can be used to detect increases, decreases, inversions or rearrangements of heterochromatic regions. The breakdown of euchromatic DNA to leave the darkly staining C-bands (heterochromatic regions) is believed to occur by a three step process:
1) depurination of the DNA by acid treatment (HCl),
2) denaturation of the DNA by alkaline treatment (barium hydroxide),
3) breakdown and removal of the denatured DNA chain at the depurinated sites by treatment with hot salts (2 x SSC).
Regions containing satellite DNA (heterochromatin) resist this degradation due either to their relatively dense packing of simple base pair repeats or to specific bound proteins.

SLIDES
Prepare microscope slides as usual. If slides are fresh try aging on a hotplate for 15mins at 75C prior to C banding.

REAGENTS / EQUIPMENT
1. 2 x heat resistant coplin jars with screw on lid
2. Barium Hydroxide [Ba(OH)2] (saturated) BDH 10048
3. 2xSSC
4. 1M HCl
5. Leishmans stain BDH 35022
6. Buffer pH 6.8 BDH Gurr Buffer tablets 33199
7. 60oC waterbath
8. Thermometer
9. 24mm x 60mm coverslip

PROCEDURE
1. To prepare for banding,
• Warm 50 ml of 2 x SSC in a Coplin jar in 60C waterbath
• Warm 50 ml of distilled water in a Coplin jar in 60C waterbath
• Weigh out 5g of Barium Hydroxide [Ba(OH)2]
2. Place slide to be banded on a staining rack and flood with 1M HCl for 90 seconds.
3. Rinse well with tap water.
4. Add Ba(OH)2 to the distilled water and stir with a thermometer until the temperature reaches 56oC - 58oC.
5. Immerse the slide in Ba(OH)2
- Blood preparations approximately 30 secs.
- Amnio or CVS preparations approximately 10 - 15 secs.
6. Rinse well with tap water.
7. Incubate in 2 x SSC at 60oC for 10 minutes.
8. Rinse and stain with 14% Leishmans (1ml Leishmans in 6ml pH 6.8 buffer) for 15 mins.

REFERENCES
1. Hoimquist G. (1979) The mechanism of C-banding; Depurination and b-elimination. Chromosoma; 72:203-224.
2. Sumner AT et al. (1971) New techniques for distinguishing between human chromosomes. Nature New Biol., 232:31-32.
3. Sumner AT. (1972) A simple technique for demonstrating centromeric heterochromatin. Exp. Cell Res., 75:304-306, modified by the Cytogenetics Laboratory, St. Vincent's Hospital, Melbourne.

NOTES and ATTACHMENTS
• If C-banding is not distinct (i.e. there is poor differentiation between euchromatin and heterochromatin) try incubating in Ba(OH)2 for an additional 5 - 10 seconds.
• If the chromosomes appear "puffed out" and completely distorted, with the associated loss of C-banding, try reducing the Ba(OH)2 time.
• The Ba(OH)2 may be used a few times, but best results are achieved with a fresh solution. Try this if results continue to be poor.

Thanks Karyotyper. I will definitely carry out your protocol. There is some variation in conc of HCl and Ba(OH)2 between both the protocols.
Can you please let me know,
(1) Will your protcol work with my slides which were made a month ago and stored at RT. I have never baked them. Do I need to bake them before C banding (even if they are not frehs) ?
(2) Do you always bake your slides before staining? I am working for teahcing lab, so when students make their slides, do I store them at RT and bake at the time of staining or bake them first and then store?
(3) Is it ok to make SSC one day in advance?
Many thanks for your time,
V

Hi again Vibha
I have no experience with slides that old. We artificially "age" our slides by heating them (as described), either shortly after preparation or the morning after, then C band them straight away. The aim of baking them is to artificially increase their "age". In the deep dark past we used to leave the slides for a few days before C banding but these days we always bake them. I don't know how long you can leave them before C banding but I would guess it would be quite some time.

You would definately not need to bake them if they are a month old (as they are already very aged). Just try the method directly on one slide and see if it works. If you have time, drop a fresh slide, bake it, and run it in parallel with the old slide.

The 2xSSC can be made anytime and is not critical. Let me know how you go (perhaps post an image if you're still having trouble and we can take it from there).

All the best.

Thanks Karyotyper for your prompt reply. I wil try it with both- old slides which are aged by storing them for 2-4wks and fresh made slides, that are aged by baking. I am eager to try your protocol and come back to you as soon as I have done it.
Thanks once again
V

HI Karyotyper,
Thanks very much for your protocol. I tried it with my aged slides ( a month old, aged without baking) and fresh slides, aged by baking at 75C for 15 min. Your protocol worked well for the baked slides but not for the first slides.
I got good C banding with most of the metaphases. But with some chromosomes, I got dark outline around each chromosome and there was overall staining.
Could you please tell me,
How to get sharp contrast of C banding, do I need to alter staining time? or stains should be more diluted? I would like to have faintly stained chromosome but very darkly stained centromere.
Many thanks for your time
Vibha.

Hi Vibha
I'm not sure I understand what you are seeing. Is part of the metaphase C-banded and part of it not C-banded? Or is it that some metaphases are C-banded and others not? Is it possible for you to post an image of what you are seeing?

HI Karyotyper,
I have no facility of taking an image.
I made a batch of 10 slides from the same culture and treated them simultaneously. However in some slides- no particular C banding was seen (all chromosomes purple all over, all metaphases) whereas some slides got C banding. When I got C banding- I observed all the chromosomes in a metaphase got C banding, whereas some metaphases in the same slide, not C banded. Contrast is ok but how to get clear contrast? I mean darkly stained centromere compared to the rest of the chromosome?
Sorry if I am confusing you
Cheers
Vibha.

Hi again Vibha
Could your hotplate temperature be variable? It seems odd that some slides show C-banding and some do not. You could try increasing the baking time to 30 mins (allow the slides to cool to room temp prior to banding). Otherwise you could leave them in a 37C incubator overnight and see if this gives a better result. You could also increase the BaOH2 another 10 or 15 secs.

The stain concentration should be fine for uniform staining (pale euchromatin, dark heterochromatin) if the method has worked OK.

If the metaphases are very cytoplasmic this might also affect the results and you will need "harsher" treatment.
Hope some of these suggestions help.
All the best.