After the formaldehyde/MgCl2 washes I washed the slides in 2X SSC and then when I put the slides in 70% ethanol the slides exude a milky cloud. Why would the slides appear milky during chromosome pretreatment before in situ hybridization?
Hello Sirjan!
Do you wash the slides in 90% and 100% ethanol after 70% ethanol?
This kind of reaction occurs when you put the slides directly into 100% ethanol after 2xSSC washing.
Regards,
Ilya
Hi Sirjan!
I don't use formaldehyde/MgCl2, but sometimes I've observed the same reaction. For this reason I do two washes in 70% and after, the wash in 95%. I think that it is a reaction between 2xSSC and the ethanol. Best, Alejandra.
Thank you all, I appreciate it! I used to do the 1X PBS after formaldehyde/Mgcl2 in PBS before doing the 2X SSC washes and had never seen the milkiness. This time I skipped the 1X PBS and directly did the 2X SSC after formaldehyde/Mgcl2 step, so my guess was having MgCl2 in SSC caused that reaction. I always do the 70%, 90% and then 100% ethanol for dehydration. Another thing different about this wash was the protocol used for chromosome preparation, I used to do the EMA method but I switched to SteamDrop method to get better spreading. Doing two 70% ethanol washes before 90% sounds like a good idea. I know some people also used chilled ethanol, do you know how effective it is and what % of ethanol is chilled to use? I appreciate the concerns!
I do not think that protocol of chromosome preparation may really influence on this kind of effect. Why you do not pretreat chromosomes by 4% paraformaldehyde buffered in 2xSSC (pH 9.0) instead of formaldehyde/Mgcl2? It is easier and preserves chromosome structure very well.
We always use chilled 70% ethanol.
Thank you Kirov, I will try that. What do you think about the pepsin treatment and its effect with that protocol? Does pepsin treatment affect the chromatin structure to a great extent? I am doing FISH with Pearlmillet (Pennisetum sp.), it has large sized chromosome (~150 Mb on average), I have been using 10 minute pepsin treatment with EMA chromosome preparation. I would appreciate your suggestions!
You can try chilled ethanol series for better result. Yes pepsin treatment will affect the chromatin structure. what you are following 10 minutes pepsin treatment is fine. Can try these steps for pre-treatment - 30 mins in 2XSSc at 37C- 0.01Hcl+ pepsin treatment for 10 minutes 37C - 1XPBS 2 minutes - formaldehyde/Mgcl2 for 5 minutes in room temp - 1XPBS 2 minutes - ethanol series 2 minutes each.
Meenakshi, thank you! I do the same washes but the time is different. I do 2X SSC washes for 3 times for 5 minutes to begin with, I do formaldehyde/MgCl2 step for 10 minutes and ethanol washes for 3 minutes. I am looking to try chilled ethanol and paraformaldehyde in 2X SSC instead of formaldehyde/MgCl2 as Kirov suggested and go from there!
Dear Sirijan!
Duration of pepsin treatment strongly depends on chromosome preparation quality. For example we do not use pepsin treatment for SteamDrop prepared slides as they are very clean normally and pepsin treatment (even 60 sec, 5 µg/ml in 0.01 M HCl) may cause chromosome damage.
Ilya
Thank you Kirov! I had used the suspension drop method without the steam, but I am amazed at your protocol. It gave me amazing result, now I can use less root tips and get way better spreads.
I will test the spreads with and without the pepsin treatment. And I Will let you know about what I get!
- Badges
- Science topic