In situ Hybridization problem with TCS solution?? - (May/17/2005 )
Has anyone any experience from running a standard ISH protocol with radiolabelled oligonucleotides of 45nt length on 30µm sections that have been in Tissue Collection Solution (TCS - glycerol, Ethelen glycol, Phosphate buffer 0.1M)
Here is the thing:
- My oligonucleotide probe works just fine on 10µm frozen sections (i.e. no TCS).
- Riboprobes of >100nt works just fine on the 30µm sections that have been in TCS.
- My oligo does not work on the 30µm TCS-treated sections.
Why is the oligonucletide not working on my sections?! I can't really understand it. The oligonucletide should have higher accessibility than the riboprobe, which is >100 bases. My oligo is only 45nt long and should easily penetrate and hybridize at 42 degr. celsius.
Anyone suggestions, comments?
Here is the thing:
- My oligonucleotide probe works just fine on 10µm frozen sections (i.e. no TCS).
- Riboprobes of >100nt works just fine on the 30µm sections that have been in TCS.
- My oligo does not work on the 30µm TCS-treated sections.
Why is the oligonucletide not working on my sections?! I can't really understand it. The oligonucletide should have higher accessibility than the riboprobe, which is >100 bases. My oligo is only 45nt long and should easily penetrate and hybridize at 42 degr. celsius.
Anyone suggestions, comments?
penetration is probably fine but I guess the probe doesn't hybridize under the same conditions as the 100bp probe does........check your hybridization conditions. try hybridizing the 45 bp at 37oC with 25%formamide/2x SSC in the hybmix. That always does it for us (50bp oligo). E.g. a 35 bp oligo doesn't hybridize at 37oC with these conditions but goes fine at roomtemp.