@dear Elske, as a quick shot I would recommend to use the (full text) search function available in RG:  try and insert the following URL (when you are logged in to RG):

https://www.researchgate.net/search.Search.html?type=question&query=fix++after+cryo++freez++IHC , 

wait just some seconds and find many Q&A, dealing with (nearly) the same matter...

best wishes and good luck, WM

Thank you Wolfgang, I have read the extensive questions posted under fixating but unfortunatly I haven't found examples of fixation after sucrose and -80C storage. Therefore a direct answer on how to proceed will come in handy - most questions deal with fixation during collection or shortly after, while I have suspended sucrose tissue which I want to fixate after. But thank you for your suggestion I will continue to search for the answer.

Best, EdH

(apologize for lengthiness....but the matter deserves....)

Good morning, good day, dear Elske, I understand and really know and feel your "pain".

Thank you for commenting on the unsuccessful search in the RG-thread-archive.

So I try to give a "direct answer" which is not quite easy since freezing a piece of tissue (even if it was frozen state-of-the-art), storing it, and finally - again - thawing it to fix the tissue by e. g. (buffered) FA-solution IMHO is rather unusual.

In addition:   It always "depends...."

First of all we should know the size of the tissue to be (post)fixed. If it is 'whole' chicken brain there will be only one or two option(s): to cool down buffered formaldehyde solution as much as possible (guessing it will be near 0°C) and placing / immersing the frozen brain into the precooled FA-solution [done in the fridge. If you can place a specimen rotator in the fridge to rotate the brain(s) during fixation this would help a lot].

Knowing this mode of "ice-cold" fixation in buffered FA (due to freezing at an early stage) might be problematic in terms of achievable morphology or also preserved antigenicity [see *) below in the cited text]  an alcoholic fixation (i. e. immersion of the deep frozen brain specs into really COLD 95%-96% EtOH) is imaginable.

"really COLD Ethanol=EtOH or Methanol MetOH [or Acetone] means that you can "cool down to at least MINUS 90°C, lower than the recrystallization point at which secondary ice crystals in biological samples are formed. The trick then will be increasing temperature until room temperature or at least 4°C. Such processing could be termed as: "progressive increasing temperature fixation=PITF" (contrary to the PLT- = progressive lowering temperature technique which is used in EM-processing/embedding into special resins for examination in TEM= transmission electron microscopy)

FYI and convenience: 95-96% EtOH has a Melting / Freezing point at: MINUS 144°C (-227.2°F);     Acetone has Freezing point at MINUS 94,7°C, (100%) Methanol : MINUS 97,68°C.

citation:   "       Frozen Tissue

One of the benefits of frozen tissue samples is the time saved by omitting the initial fixation step required for paraffin-embedded tissue. Snap freezing can be particularly beneficial when detecting post-translational modifications such as phosphorylation. Frozen tissue can be prepared by immersing the tissue in liquid nitrogen or isopentane, or by burying it in dry ice. For frozen samples, a short fixation is done after freezing and sectioning.     [ *)] Frozen tissue sections are most often fixed using alcohol, which avoids the possible requirement to retrieve epitopes masked by formaldehyde cross-linking  [ *)] . Frozen tissue is cut on a cryostat and sections can be stored at -80 °C for up to 1 year.  ......   .....  Because frozen tissue often retains enzymatic activities, blocking the functions of endogenous enzymes that may affect the IHC detection method is also particularly important."

[cited from: https://www.rndsystems.com/resources/protocols/preparing-samples-ihcicc-experiments]

Also mixture of ice cold acetone or / and ice cold Methanol (freezing points see above)  could be considered to be a possibility  ["Water in the samples is replaced by a solvent (usually acetone) at a temperature around −90 °C. This temperature is lower than the lowest temperature at which secondary ice crystals have been shown to form in biological samples (−70 °C) (Steinbrecht, 1985)," citation from: cf. http://www.sciencedirect.com/science/article/pii/S0378595514001178 ]

but regarding acetone or methanol, also cf:  https://www.researchgate.net/post/Solution_in_IF_staining and  - perhaps very informative -  HUANG & YEUNG,_Chemical&Physical fixation of Cells&Tissues_an overview, Chapter 2 p. 23ff,, pp.24/25  © Springer International Publishing Switzerland 2015 23

E. C. T. Yeung et al. (eds.), Plant Microtechniques and Protocols,

DOI 10.1007/978-3-319-19944-3_2 [= cf: http://www.springer.com/cda/content/document/cda_downloaddocument/9783319199436-c1.pdf]

The (most possibly impractical) other approach:  cryosectioning thicker sections (25-50µm) and immersing those sections immediately into ice cold buffered FA-solution. then processing as usual into Paraffin.

Perhaps use of Microwave oven (supporting fixation) could help / facilitate the difficile task.

(cf. e.g.: https://www.jstage.jst.go.jp/article/ahc1968/23/4/23_4_573/_pdf )

The problem - when 'thawing and fixing' (which happens physically in a setting as described above) tissue blocks/organs too big in size is going beyond the recrystallization temperature (at minus 80°C) which leads most possibly to a formation of ice crystals. Such may negatively affect tissue structure as well as uneven or flawed presentation of antigenic sites you would like to localize afterwards.

If "fixing" only by 95-96% 'icecold' EtOH the problem [besides poor preservation of texture] eventually will be the water solubility of components which were NOT fixed - at least after paraffin embedding - when staining sections.

NB: During my former work I have done / had to process three or four times frozen tissue specimens for embedding int resin for "ultrastructural" evaluation /examination. Needless to say that ultratsructural morphology was poor (mainly due also to poor freezing method(s) used. Using the "ice cold" buffered FA-solution technique (= as cold as possible without freezing of the solution) and slow(!) thawing of the whole system in the fridge it was possible to achieve at least acceptable 'morphology' in semithin (=µm 'thick') plastic resin sections. It therefore it can be argued that histomorphological appearance could be acceptable after such processing. I would not recommend to remove the sucrose / saccharose in the frozen tissue block with "cold"  ( phosphate or any other ) buffer first....

Again, apologize for lengthiness... but the matter is tricky and needs compromises...Nevertheless I hope you enjoy reading and find it helpful.

Best wishes and regards, Wolfgang

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Further reading (with regard to rapid freezing physics-not exhausting-):  

http://www.sciencedirect.com/science/article/pii/S0378595514001178

http://www.feinberg.northwestern.edu/research/docs/cores/mhpl/tissuefreezing.pdf

http://www.jefferson.edu/content/dam/skcc/Sample%20prep-frozen%20embedding.pdf

http://www.phenogenomics.ca/docs/PathologySamplePrepGuide_20150315.pdf

http://aem.asm.org/content/72/2/1702.full

https://www.jstage.jst.go.jp/article/ahc1968/23/4/23_4_573/_pdf

Dear Wolfgang, a very good afternoon to you.

So nice that you took this effort to inform me about all the possibilities and risks of this post fixing problem of mine. I enjoyed reading the text and I will consider the different options. But as you say, the ice cold FA with slow thawing might give me reasonable morphology. I will give it a try with some tissue I can afford to loose. Thanks so much,

Elske

Dear Elske, you are welcome (:-))