In situ hybridization performed on rat liver sections, paraffin - Does anyone have good cellular morphology with NBT/BCIP? (Mar/25/2008 )

Hi everyone!

I'm having some difficulty with maintaining good cellular morphology in the rat liver after in situ hybridization using NBT/BCIP colour reaction. Our resident pathologists say that the cells are not looking very happy at 60X magnification light microscopy.

I was wanted to know if anyone has had experience with in situ in this tissue and how they maintain good cellular integrity. I've been using an in situ protocol that works perfectly on brain sections but adapting it to the liver has been troublesome. I do not use proteinase K, at most I use Tween 20 in my solutions. I think it is the NBT/BCIP precipitates themselves or perhaps the colour reactions solution.

I would appreciate any suggestions!

Sincerely,

D

Hi,

I am not familiar with NBT/BCIP or mice tissue, but I do In situ quite often.
anyway, mostlikely you cannot use the same protocol to different section/tissue.
I do FISH as well as In situ with HRP-DAB for human section (paraffin) like brain, liver, pancreas, lung, skin or so, but I always need to modify the protocol depending on the tissues. (because different tissue has different integrity)

also, you have to consider the fixation step. Some tissue might be fixed in PFA for longer or shorter time, which affect histology result, and sometimes age of block affect results too.

to be honest, In situ hybridization is one of the greatest methods, but most frastrating methods to optimize for me.

anyway I hope this helps

rnotk

Hi rnotk,

What parts of the protocol do you modify for the tissue with finicky cellular integrity? Do you find that the liver is sensitive to in situs? Basically, for the brain under light microscopy, even at 60X the cells look intact with distinct staining. In the liver, however, the cells look sickly and the stain takes longer to come up and affects the intra-cellular structure (even at 40X).

I'm using the roche anti-dig antibodies right now (the AP antibody is very good), but I would be interested in an anti-dig HRP that works well (I'm not confident in the roche anti-dig HRP) if you could recommend one.

I've thought about the period of time it's been in fixative (formalin), but the Hemotoxylin and Eosin stains show that the sections look fine.

well, nomally I will change the retreival process,
like, retreival solution (I usually use DAKO citrate based retreival) in microwave retreival at 90-95C for 8-20min
I also do proteinase K digestion after that (8-10min either RT or 37C).
(I use 4-6um section)
our probe is pre-labeled with fluorescent tag for FISH, but I also make tag by myself so it will have DIG label,
in this canse, I use anti-DIG-HRP antibody, then DAB
Liver is extra fatty compared to the other tissue, so I normally do longer Pro K, but if morphology is lost for your section,
I think that I saw some protocol that is refixed in middle of the In situ process using PFA, although I dont do this, so I dont have detail protocol

rnotk

Dear rnotk,

which company do you purchase you anti-dig HRP from?

sorry for the late reply
Our Univ was Spring brake last week, and I was staying my home to write paper....

Anyway, I use to use Rabbit anti Dig-HRP from DAKO, but they dont have one anymore, so
I now use mouse anti Dig HRP from abcam, and work well on paraffin section.

Rnotk

Why do you think that the NBT/BCIP colour reaction is the reason for the changed morphology? Bad fixation or too high hybridization temperatures seem to be more likely the reason for that. You should try to fix a bit longer and/or try a lower hybridization temperature. Normally Formamid is added to the Hyb-Buffer so theoretically you could try to do the hybridization at around 60°C.