Paraformaldehyde-fixed frozen tissue

AR by Heating En Bloc for Paraformaldehyde -Fixed Frozen Tissue... 

Gill et al.21 Archival FFPE spinal cord tissue both paraformaldehyde-fixed frozen rat spinal cord tissue and paraffin-embedded same tissue To establish an optimal protocol for detection of low-abundance protein (NeuN) in human spinal cord FFPE tissue sections, testing three AR solutions of pH 6, alkaline, and acidic buffer, with three heating conditions 95,100, and 105°C Heating FFPE tissue sections in an alkaline buffer yields most effective AR-IHC staining results. 

Archival FFPE spinal cord tissue both paraformaldehyde-fixed frozen rat spinal cord tissue and paraffin-embedded same tissue... 

Here we present two protocols developed to investigate the spatial distribution and relationship of neuro active substances in the nervous tissues of cephalopod molluscs. The protocols are designed for frozen and vibratome sections of the Octopus vuljfarishiain, but are easily transferable to other cephalopod species and tissues, and are also specifically designed to detect small molecules such as monoamines. One of the two protocols has been adjusted to process paraformaldehyde-fixed tissues, while the second is designed for tissues that require glutaraldehyde frxation. 

Frozen material or tissue culture cells may be fixed by immersion for 10 min in 50% methanol/50% acetone at -20°C, followed by air drying. Alternatively, a 4% solution of paraformaldehyde can be prepared by dissolving 4 g of paraformaldehyde powder in 80 mL of water with gentle heating and the addition of 1 M NaOH until the powder dissolves. The solution is made up to 90 mL with distilled water, and 10 mL of a 10X PBS solution added. 

We found that fresh, unfixed tissue from the frozen brains showed a substantially stronger reaction for both stains than tissue fixed with formalin, paraformaldehyde, glutaraldehyde, or alcohol. 

This new definition of immunocytochemistry derives from advances in antibodylabeling methods in recent years. These advances resulted from specific needs in animal research. Initially, formalin-fixed paraffin sections were used for immuno-histochemistry however, results were inconsistent. In most cases, the antibody did not label anything or it labeled too many cells and was dubbed over fixed. This problem led to the development of the epitope retrieval or antigen retrieval methods, where sections of tissue are treated with heat in buffers before antibody incubations. Unfortunately, epitope retrieval methods can be unique from antibody to antibody and also, for the same antibody, from tissue to tissue. Epitope retrieval is complicated and best avoided. For animal research, a simple method was then developed where tissue was fixed in paraformaldehyde and not formalin or alcohol and subsequently frozen sections were cut on a cryostat. This eliminated the steps of dehydration, embedding in paraffin, rehydration after sectioning, and epitope retrieval before antibody incubation. This was a major breakthrough. 

Frozen fixed tissue is sectioned in a cryostat also known as a microtome in a freezer. The tissue is fixed in 4% paraformaldehyde, rinsed, and then cryoprotected by infiltration in 20% sucrose in buffer with agitation overnight at 4°C (cold room). After 24 h the tissue blocks will sink in the solution, indicating that they are infiltrated. This infiltration step is critical. If skipped, the tissue will freeze with damaged cells and holes from ice crystals, and the resulting tissue sections on microscope slides will be brittle and might crack. The tissue must be fixed first to hold the cells in place, as cryoprotection and freezing without fixation will destroy the tissue. 

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