Dear Michael, I do not understand what you intend with "scramble". Do you have a high fluorescent signal also in controls? Please let me know more in detail your protocol and I try to give you some suggestion. 70 degrees is a very high temp. I perform my ISH at 55°C.

Barbara

Hi  Barbara,

The scramble is a scramble sequence which is predicted by blast not to hybridize to any RNA. Thus it acts as a negative control and I shouldn't see anything in this condition but it's very bright. My hybridization has started out at 42 degrees but I've been raising the temperature in an attempt to decrease partial hybridization of the pore to other RNA species.  Thanks for your help.

What are your sections fixed in ?? PFA tends to give a lot of background which looks as good as the positive staining. I have not been able to get rid of it especially if it is the FITC/GFP channel. Although there are protocols to quench the autoflourescence, they dont seem o work in my hands for PFA fixed samples. 

I would suggest to try an alternate fixative.

As barbra suggested for a 52 mer 70C is very high a temperature, especially if you are using  formamide (unless its a ribo) 

Is it tail labelled or end labeled probe ? 

If its tail labeled, include poly A in the hyb mix, as at times the tail loves to stick to repeats.

Hope this helps  

Thanks Deepak. I am using 3% PFA in PBS for 20 minutes on cultured neurons. What type of fixative would you suggest? I never really had issues before with autofluorescence from PFA in our culture model but I'm willing to try anything!

I agree 70C is high. My protocol had initially 42c as a hybridization temp but I've been increasing the temp in order to increase the stringency of the reaction. The signal of the scramble has been diminishing with greater temps but is still too high. My probes are end-labelled.

Dear Michael. You tried to increase the stringency washes? After  0.2 X SSC washes, could add a wash of 15% formamide at 42 ° C for 20 min, and increase the concentration of  salmon sperm DNA + yeast tRNA. 

Thanks Jonathan! That was my next step. Also thinking of increasing formamide concentration in my hybridization buffer from 50% to 70%.

Hello Michael,

I have been doing RNA FISH successfully for several years.  I have not worked with neuronal cells but have worked with ESCs.  First of all, try using COT-1 DNA in your hybridization buffer to quench hybridization of repetitive sequences.  (Is it possible that your scramble probe has a repetitive sequence?). Secondly, I wash my slides 1X 50% Formamide @ ~40° C. In preparing my slides, I pretreat with an extraction step of  ice cold CSK buffer/0.5% TX-100/RVC for 10 minutes.  I then fix my slides with 4% paraformaldehyde.  I store them @ -20° C in 70% ethanol until needed.  I have found that my slides work have less background if I let them age for 1-2days prior to hybridization. I have attached a complete protocol for your reference.   

There is a picture of Xist RNA FISH on my personal page showing a typical signal.  

Thank you Leslie! My sequence does not have any repetitive sequence but I will try aging the cells in 70%. Good tip!

Sorry Michael, Realized it now, do you get signals in your no probe controls ? This needs to be ruled out to see if its autoflourescence or issue of mis hybridization. IF your no probe controls are clean then work on the hyb or rather washings !  

Have you tried sense sequences instead of scrambled. I believe they are more ideal for ISH as negative controls than the so called scrambled, 

PS: Methanol or acetone alone are also good fixatives to reduce autoflourescence. 

Hi Michael. I have been doing FISH for some years in PFA-fixed cell tissue, but not in neuronal cultures. My experience tells me that: first of all it is recommended to check the specificity of the primary antibody (since I had some trouble with my antibody).

Second, maybe is better to use as a control the sense probe rather than the scramble probe.

Third, you can try to dilute your probe (maybe the concentration is too high and stringency washes cannot clean all the unbound probe). The same with the antibody.

I do not know if, in the post-hybridization, you wash with RNAse A. If not, maybe is a good call in order to remove all the unbound probe.

If you want my protocol for FISH let me know and I´ll send to you.

Good luck Michael! 

Thank you Juan.

i am using fluorescently tagged DNA oligonucleotides so there is no antibody involved. We thought about using a sense strand but my mRNA has complementary sequences on the other end making it hard to develop a sense strand as a negative control. Getting rid of the unbound single stranded DNA probe enzymatically sounds like something that could work! Thank you. Can you send me your protocol please?

Thank you Deepak,

Will give it a try.

Michael