Hello Group!
I fixed the cells with carnoy's fixative and cause the metaphase spread on the slide and stain it with Giemsa two weeks ago.
Now I want to destain the slide and stain the centromere by FISH. I destained it with 100% methanol for 5 mins. After drying, I add 5ul centromere Enumeration Probe mixture and cover with coverslip 22x22 mm^2 and seal with rubber cement. Denature sample and probe by heating the slide on a hot plate at 75 degree for 2mins. Then incubate it in humidified chamber in 37 degree for 2 hours.After that remove the coverslip, wash slide by 0.4xSSC in 72 degree for 2mins. Then wash by 2xSSC, 0.05% Tween-20 in RT. rinse briefly by aqua dest. Apply 10ul DAPI/antifade and cover the coverslip for 10mins.
But there is no green signal of centromere detected in DAPI mode in florescence microscope. I only can see the whole chromosome in blue.
May I know if any mistakes in my protocol?
Thanks a lot!
https://www.researchgate.net/post/Techniques_to_destain_Giemsa-stained_cell_colonies
Maybe you can try some of the methods Wolfgang H. Muss recomended in this post.
but for my experience if I´am thinking to destain something I for sure will avoid Carnoy on the first step.
Giemsa can be removed with Carnoy solution (methanol based, once Giemsa is ). Use it in a coplin jar for 5 to 10 min. The special problem to do FISH after Giemsa actually will be the immersion oil. This Carnoy bath can remove some, but if your chromosomes are resistent to xylene, maybe is good to use it to remove the oil.
You can remove Giemsa stain by simply putting your slide in xylene for 1 or 2 days depending upon how much dark the stain is. It will remove the coverslip, extra dpx as well as extra stain from slide. if the stain still remains on slide try to put the slide in lukewarm water for few minutes. This process usually destain the slide but if still you face problem then you can wash it in buffer solution. it will really help.
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