Destaining FISH slide and successful G-Banding protocol

[Santosh Rani]

Dear group members,  Request if there is any specific procedure wherein we can destain DAPI slides post FISH analysis and be successful at getting good G-Banding results on a metaphase found by FISH to be abnormal. We are having issues with cells resisting trypsin. Appreciate if any of the group members can share protocol and thoughts on this. Regards, Santosh

[sd]

Hello,

 I used twice the procedure described in this paper with good results.

http://onlinelibrary.wiley.com/doi/10.1002/cyto.990210410/epdf

Hope this help,

Stella

[Santosh Rani]

Thank you Stella. Appreciate 

Sent from my iPhone

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[maria mouriki]

Hello Stella,

I read the protocol above and I have a few questions for you.

1. Have you tried the protocol on interphase and metaphase FISH?

2. Have you tried the protocol using centromeric and locus specific probes?

3. The 3 washes of the slide in 4xSSC/0.1% Triton X-100 were for 2 minutes each?

4. How did you do the dehydration step of the slides in graded ethanol: did you use 70%-85%-100% ethanol for 2 minutes each?

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Μουρίκη Μαρία

Κλινική Κυτταρογενετίστρια

UK Health and Care Professions Council Certified

DNA BIOLAB

2810 323034

[sd]

Hello Maria, 

I used this protocol on interphase FISH, using LSI probes. For the dehydration step i used 70%-85%-100% ethanol for 2 minutes each as you mention. 

As for question 3, I am really sorry but i am out of office up until 4/9/2017. It will be my pleasure to respond to all of your inquiries by then if this in not a big issue for you. 

S

 

On Wednesday, June 21, 2017 at 12:55:38 PM UTC+3, Santosh Rani wrote:

[sd]

Hello Maria, 

I am looking at my notes for the destaining protocol. i used a washing solution  0,4XSSC/0,3% NP-40 for two minutes. 

Hope this help.

S

On Wednesday, August 23, 2017 at 1:43:07 PM UTC+3, Maria Mouriki wrote:

Hello Stella,

I read the protocol above and I have a few questions for you.

1. Have you tried the protocol on interphase and metaphase FISH?

2. Have you tried the protocol using centromeric and locus specific probes?

3. The 3 washes of the slide in 4xSSC/0.1% Triton X-100 were for 2 minutes each?

4. How did you do the dehydration step of the slides in graded ethanol: did you use 70%-85%-100% ethanol for 2 minutes each?

[maria mouriki]

Goodmorning Stella! Thank you very much for your response. I will keep your protocol in my notes for future reference. Have a nice day!

On Mon, Sep 4, 2017 at 9:54 AM, 'sd' via Cytogenetics methods and trouble-shooting Forum <cytogenetics-methods...@googlegroups.com> wrote:

Hello Maria, 

I am looking at my notes for the destaining protocol. i used a washing solution  0,4XSSC/0,3% NP-40 for two minutes. 

Hope this help.

S

On Wednesday, August 23, 2017 at 1:43:07 PM UTC+3, Maria Mouriki wrote:

Hello Stella,

I read the protocol above and I have a few questions for you.

1. Have you tried the protocol on interphase and metaphase FISH?

2. Have you tried the protocol using centromeric and locus specific probes?

3. The 3 washes of the slide in 4xSSC/0.1% Triton X-100 were for 2 minutes each?

4. How did you do the dehydration step of the slides in graded ethanol: did you use 70%-85%-100% ethanol for 2 minutes each?

On Tue, Aug 8, 2017 at 2:13 PM, 'sd' via Cytogenetics methods and trouble-shooting Forum <cytogenetics-methods-and-trouble-sh...@googlegroups.com> wrote:

Hello,

 I used twice the procedure described in this paper with good results.

http://onlinelibrary.wiley.com/doi/10.1002/cyto.990210410/epdf

Hope this help,

Stella

On Wednesday, June 21, 2017 at 12:55:38 PM UTC+3, Santosh Rani wrote:

Dear group members,  Request if there is any specific procedure wherein we can destain DAPI slides post FISH analysis and be successful at getting good G-Banding results on a metaphase found by FISH to be abnormal. We are having issues with cells resisting trypsin. Appreciate if any of the group members can share protocol and thoughts on this. Regards, Santosh

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Μουρίκη Μαρία

Κλινική Κυτταρογενετίστρια

UK Health and Care Professions Council Certified

DNA BIOLAB

2810 323034

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