Dear Nisha,

good question. The standard textbook answer would be "no - it cannot". I tried it myself a while back and it didn't work - the non-specific binding was huge and the image quality very poor compared to our fresh frozen tissue from the same organs  

However, I reviewed a paper recently in which the authors had used deparaffinized brain tissue sections with success. So I guess it can be done. Cannot give you any details or references yet, as that paper is not yet published. It will be soon I think.

Maybe other researchers can give you a clue as to how to do it. 

Bw Per

Dear Nisha,

My guess is that it will not work when paraffin embedded sections are directly used since the paraffin will prevent the ligand from entering the sections, and the PET probe may bind nonspecifically to the paraffin. However, I have heard that paraffin can be removed from embedded tissue sections (by treating them with an appropriate solvent?) and perhaps, after removal of paraffin the sections may still be useful for autoradiography. Anyhow, such procedures would require an investigator that has considerably more manual skills than clumsy me !

You may perhaps check the literature and find out the proper procedure for removal of paraffin. Also, you could check if anything has been written about the possible uses of de-paraffinized tissue sections, e.g. if they have been successfully incubated with tritiated or 14C-labeled tracers (more references may be available about such tracers than about PET ligands).

One person who may know is professor Kubota from Japan who is an expert in (micro) autoradiography.

Best regards,

Aren

Dear Per and Aren,

Thank you for your replies. We gave it a try anyway and it was fun to watch the liquids behaving like mercury on top of the slides and managed to get black blobs for our trouble! I will look into deparaffinizing.

Best Regards,

Nisha

Dear Nisha, I have no experiance with PET ligands, but in former times I worked with  tritiated Proline in paraffin  and in frozen ssections. In both cases we got good results. If you are working with paraffin sections it is necessary to dewax the paraffin sectios before you start with the autoradiography. Here is the deparaffination protocol.

Xylene I - over night; Xylene II-IV - 15 minutes each; let sections dry out after the last Xylene;  100% EtOH or Isoprpanol - 15 Min; 96 % EtOH, 70 % ETOH, 50 % EtOH - 15 min each, destilled water - 15 min;  dry the sections between 30 and 35 °C in the oven. Cover your sections with X-ray films or with foto emulsion. The exposure time depends on the kind of beams and of the thicknes of your sections. If PET ligands have the same ability like tritium then you can say the thinner the sectiosn the longer the exposure. We needed for our 5 µm sections around 6 weeks  and fore30 µm frozen sectoins around 3 weeksn exposure time. Depending on your experience with frozen sections I would recommand to develop sections after  different periods of time to find out which exposure time is the best.  Hope it will help you. Good luck and best wishes, Ute

Dear Ute,

Thank you very much for the protocol. What are the different Xylene numbers? Are they different concentrations?

Regards,

Nisha

Xylene I - over night; Xylene II-IV - 15 min each describes the steps needed to "dewax" the paraffin sections (usually this is performed in a suited cuvette immersing the slides with the sections into the xylene,-  rather unspectacular and routine steps in Histology - Labs:

xylene (1st "bath") = over night

xylene II-IV=

xylene II (2nd bath, 15 min)

xylene III (3rd bath= 15 min)

xylene IV=(4th bath= 15 min)

Purity of xylene fluid will be compromised usually in baths I-III, but should be minimal if not absent (when practically all paraffin has been solved from / out of the section) in the last step of deparaffinization/dewaxing (:-))

Hi Wolfgang,

thank you for explaining the protocol. Of course this is routin work, but for those who never worked in a histol lab it is strange. Sorry Nisha, that I didn't explaine it exactly.

Now I hope that these protocol will help you really. Good luck!

Thank you Wolfgang and Ute. It is now clear to me that it is the same concentration of Xylene, but in separate containers to progressively remove the paraffin. Please excuse my ignorance of the in vitro world, I am just beginning to dip my toe in.