Hi

This article is giving a reprocessing method used on renal tumour tissue

Might help

Dear Shibabrata,

the question seems not to attract many (perhaps because your request wasn't quite detailed or precise). There are several recipes or schedules (Googleing)  (cf. "reembedding from paraffin [also FFPE] blocks or sections] and examination by [T]EM, etc.).  

For me to answer I would like to know first: your equipment you have at hand(concerning processing for and examining by EM). Do you have at least basic knowledge of EM-processing? What is the task of your EM-study/examination of the FFPE-specimens (from block or from sections?...

You see there are some parameters one must consider for a correct and/or useful answer, since "all depends"....

Otherwise my answer is e.g. for a correlative examination of structure(s) only (naturally with deduction in quality of [ultra-]structural detail):

Save a "last" H&E-section on slide, by microscopical evaluation of that section choose the small area (usually not more than 2x2mm, some can do it for 5x4 mm or even larger) you want to examine correlativly [i.e. ONLY for structures) in the TEM. If you want to perhaps localize proteins etc. by immuno-methods the following might NOT apply as described.

i1) cut out (best in an oriented fashion, either with a scalpel or using a biopsy punch 2-4 mm diameter) small pieces (from your FFPE block) of about 1 - 2 mm³ [3-5 mm³). There are several methods to facilitate that acquiring the right specimen out of the FFPE-block.

ii1) Deparaffinize (!long enough!) the small specimen you got either with xylene, or (usually now banned in labs) toluene,

iii1) go back to the water phase ("rehydration" from xylene/toluene - 100% alcohol down  (96,90,80%, resp.) to at least 70% (better also a short 50% step too) alcohol  - finally [e.g. phosphate-buffer - NOT PBS - as used in T-EM),

iv1) then perhaps "postfixation" for "EM" as usual with buffered glutaraldehyde solution, then washings in buffer solution,

v1) incubation in buffered OsO4 solution (as usually done for TEM, CAUTION: OSO4 is a very toxic & hazardous stuff which has to be handled accordingly with some personal safety regulations),

vi)1 again dehydrate and embed into resin of your choice .

vii1) After polymerization of the resin blocks follow the usual routine steps (sectioning for LM-semithin section(s) -staining for LM, then choose the area you want to examine by TEM, trim your resin block, cut ultrathin sections and mount them to , stain at least with the known double staining method (UO2Ac / Lead Citrate).

OR: after i1) above,

ii2), use  a mixture of xylene  or toluene containing at least 0.1% OsO4 (OsO4 crystal will dissolve much better than in water, but will be reduced to OsO2 also much faster. Again, cautions with handling and personal safety action neccessary! e.g. use always fume cupboard) . You can apply also some heat to the solution (e.g. at 30°C, all done under a hood!). Use specimen rotator and small glasses (specimen vials) with snap cap lid. Be sure that all paraffin wax has been dissolved from the specimen (the solution will darken due to black precipitates of OsO2), and then proceed as follows:

iii2) pure xylene or toluene 3-4 times to get rid of any unbound OsO4 from the former solution, change to organic solvents like e. g. Acetonitrile, or Acetone, respectively,

iv2) either use an organic solution like Acetonitrile, Acetone or even toluene (all p.A.! waterfree)  and mix  the resin (usually epoxide) 1:1 with the solvent.

v) Infiltrate your small specs in resin 1:1(organic solvent) for at least 1 h, using a specimen rotator, then change to pure resin at least 3-4 times for at least 45 min at RT. The latter should not be done in the glass vials you infiltrated your specs in 1:1 mixture but use other, uncontaminated glassware or plastic caps for this task. Use of a glow-light bulb some 15 to 20 cm above the specs in the infiltrating cups will aid in getting rid of the solvent out of the resin. 

vi2) embed in fresh pure resin, polymerize, etc.,etc.

This approach appropriate only for a correlative structural examination with reduced preservation of ultrastructural morphology. The latter naturally depends also on the initial fixation protocol of your FFPE-blocks as well as the processing into paraffin wax.

If you have specific questions you can get more detailed answers by writing again.

Just to add another reference:

Sibylle Widéhn, and Lars-Gunnar Kindblom:  A Rapid and Simple Method for Electron Microscopy of Paraffjm-EmbeddedTissue, Ultrastructural Pathology,12: 131 - 136, 1988 (C) by Hemisphere Publishing Corporation.

Best of luck,WM.