Hi everyone.
I am facing trouble in getting adipose tissue cryosections harvested from obese mouse. After I harvest, I fixed the samples in 4% Formaldehyde for 24 hours, after that i make cyroblock in OCT and try to get sections at 10um, 20um, 30um, in all condition i failed to get. All the tissue seems attach to blades possibly because of oily nature. The maximum temperature that i can decrease in my machine is -23C. Can anyone suggest me better protocol?
Regards
Hi,
It is very tough to cryostection adipose tissue. According to your protocol, I'm not surprised that you had a hard time to get those tissues sliced.
-Firstly, you need to cryoprotect your samples using sucrose (10% then 30%) incubation before embedding in OCT (this step also requires a particular attention. How do you embed your tissues?);
-secondly, your cryostat have to be much colder than -23'C (at least -35'C)
-and finally, it seems to me that 10, 20 or 30um is a way too thick (what is the purpose of your study? regular staining, IHC, IF?).
I would definitely recommand paraffin embedding for adipose tissue. (formal fresh O/N at 4'C, then increase EtOH stepwise from 70 to 100% and finaly 100% xylene).
If your samples are really precious, the last thing I would try is to use a cryospray to cool the cryoblock, specimen holder, blade.... to get them colder than -23'C (but since the cryospray is around -75'C, there is no efficient way to cool your block down at a defined temperature).
Best,
Dear Wattez,
Thank you for your nice suggestion. The purpose of my experiment is to do Oil O Red staining and to second to trace delivered fluorescent labelled siRNA (that is only possible with cryosection). The difficult point is i can't decrease temperature(model is too old). Can you please detail protocol for sucrose incubation, I mean how long, temperature condition etc.
Regards
Determination (coloring) of lipids on paraffin cuts is not possible! Fats will dissolve during processing !!!! (Xylene, alcohol, paraffin 60 C). Only cryo-cuts! Temperature -25 to -35 ° C.Try cutting thin sections 5um. Cryospray super help! :-)
Hi,
indeed, paraffin embedding is not suitable for ORO staining.
So after fixing your tissues with 4%PFA, remove excess PFA with PBS rinse, cryoprotect them with 10% sucrose in phosphate buffer (0.2M) then 30% sucrose (this cryoprotection by sucrose infiltration is function of tissue density and structure. It takes typically between 2-5h at 4'C).
For embedding, fill a container with dry ice and immerge a beaker containing 2-methyl butane (isopentane). Remove excess sucrose from tissue by blotting on Kimwipes and place tissue in center of a mold filled with OTC. Orient tissue into the bottom of the mold and freeze by floating on isopentane bath ( ! do not get isopentane on the OTC ! ). Blocks are ready to be stored at -80'C or sliced after they are frozen completely.
Since your cryostat does not go below -23'C, use a cryospray (tetrafluoroethane) to cool down your block and blade. This way, you should be able to get some good slides for your experiments.
Best,
I've had good luck freezing fresh tissue without sucrose infiltration. As others have suggested make your tissue block as cold as possible. You will be able to get one or two good sections before the friction of the knife begins to warm up the block. You'll have to keep getting it cold depending on how many sections you need. 10 micron sections work well for Oil Red O stain in my experience.
@Jean Wattez: I'm surprised why it is not advisable to get OCT in contact with isopentane?
Concerning the original question: I agree with the others that you have to pay attention for not using solvents that solve fat if you want to see fat... Have you tried completely unfixed cryosections?