I am currently working on human peripheral blood karyotype and having a problem which chromosomes are swollen and do not show any banding pattern. (see attached image)
After finishing harvest and slide - making procedure, we bake slide in the oven at 70 celcius degree overnight for aging chromosomes.
And this is the staining procedure we used:
- Put the slide in Trypsin coplin incubating 30 celcius degree for 5 - 10 seconds
- Rinse the slide twice with 2 coplin of PBS
- Stain the slide in Giemsa 2.5% for 2 minutes
- Rinse the slide in the stream of water and air dry
We used PBS pH 7.4 (NaCl, KCl, Na2HPO4, KH2PO4/ in injected water) for trypsin preparetion and rinsing slides, phosphate buffer pH 6.8 (Na2HPO4, NaH2PO4/ in injected water) for Giemsa working
At first, we thought that swollen chromosome can be reason by failure in aging slide, we tried others (90 celcius degree for 1h/2h/4h, room temperature for several days, or aging on slide warmer 75 celcius degree for 2h) but did not make the slide better.
Does anyone have experience for this problem?
The chromosomes that you are presenting in the photo are absolutely digested by trypsin. In other words, they are over exposed to the action of trypsin. We have good results: we incubate the slides in the oven for one hour(100 centigrades), put the slides in the trypsin solution (0.125 gr / in 10 ml of buffer 6.8 at 37 degrees) for 10 to 15 seconds (this time could vary according to the environmental conditions of humidity and temperature)
after these steps we do the staining of slides with giemsa at 10%
luis alberto mendez-rosado I used trysin EDTA solution 0.5% (stock 10X) and dilute to 1X by PBS pH7.4. On my calculation, there is 0.005g Trypsin/ 10ml trypsin working solution (lower concetration than what you suggest), I tried slide with 3 second or even 1 second, some chromosome got swollen too
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