• Paraffin, as well as most of other embedding media, is hydrophobic so water must be completely removed from tissue before embedding. Tissue with water cannot be securely held in place in the hydrophobic media.
• Dehydration is the branching point to all other tissue investigation techniques: after dehydration, the tissue can be used for plastic embedding for both light and electron microscopy, or for the standard paraffin technique.

• Dehydration is usually done RT. higher temperature may be used to increase the speed of dehydration. sometimes, a lower temperature (4C) is used to decrease the extraction of some cell components for immunolocalization or in situ hybridization experiments
• water is gradually replaced by an organic solvent that is usually alcohol or acetone
• change solutions by decanting the liquid when feasible or by withdrawing the liquid with a pipette

• Tissue usually first undergoes a wash to remove fixative from tissue; the washing solution=fixative solution-the fixative
• dehydration begins with a water concentration equal to that of the fixative (use 50% EtOH for FAA because H2O in FAA~50%)
• tissue is processed through a graded dehydration series of increasing concentrations of the organic solvent
• Dehydration renders tissue transparent, the tissue is stained with 0.1%(w/v) safranin O, Eosin Y, or Thymol Blue in the penultimate 100% EtOH or acetone step in order to see the tissue in the blocks of paraffin.

%EtOH                    time

30                          1h

50                          1h

70                          1h

90                          1h

95                          1h

100+dye                 2-4h

100                         1h

An example of 100% EtOH+dye:0.1% safranin in 100% EtOH

• it is not essential to remove the solution to the last drop in every EtOH step. In fact, working too long to remove the solution might let the tissue dry out in the air, which causes irreparable damages.
• length of time in each solvent depends largely on the size and texture of the tissue;small and soft tissues need 0.5-2 hours/step, whereas large or hard tissues (e.g. wood or seeds) may need 24 hours/step
• the solutionsof dehydration (and of later steps) are organic solvents that will dissolve the glue that sticks tags and ink, so use small, paper tags marked in pencil and place the tags in the vials with the tissue specimen
• the dehydration process can be stopped for days once the tissue is in 70% or higher concentrations of EtOH

• Paraffin or plastics are used in filtration and subsequent embedding
• choice of paraffin:we will use paraplast that melts at 56C

• before being embedded, the dehydrated tisuue should be gradually infiltrated with the embedding medium, which requires a solvent to dissolve the embedding medium to various concentrations
• an intermediate solvent is used when the dehyrationn solvent is not the solvent of the embedding media, which is always the case for paraffin embedding
• Xylenne is typically used as the intermediate solvents for paraffin embedding, but others can also be used depending on the circumstances and preferences
• dehydration steps for plastic embedding overall are similar to paraffin eembedding, but nno inntermediate solvent is needed.

• after dehydrationn with EtOH, the tissues must be transferred into three mixtures(3:1, 1:1, 1:3) of EtOH and another solvent that dissolves paraffin, which can be xylene (or TBA, Histo-clear,etc) this paraffin-dissolving solvennnt is called intermediate solvent
• two changes of 100% xylene then follow the last mixture of EtOH and xylene
• when done manually, paraffin will be added in increments to last 100% xylene at room temperature, at 42C and at 58C in long incubation times; samples should be infiltrated with several changes of liquid paraffin to complete the infilittation process

• methacrylates are the basic components of plastic embedding media for LM (0.5 a few µm sections) and EM (50-70nm sections)
• a plastic medium provides a denser support matrix than paraffin, better retains a cell and tissue structure, and allows thinner sections
• tissues are infiltrated by the liquid monomeric form of methacrylates and subsequently hardened by polymerizing the methacrylates using heat, catalysts, or UV irradiation
• monomeric metthacrylates can be mixed with EtOH so that no intermediate solvent is needed

• after the last 100% xylene, the sample is immediately infiltrated with pure liquid paraffin

Solution            Duration

70%EtOH          1h

90%EtOH          1h

95%EtOH          1h

100%EtOH+dye 4h

100%EtOH         1h

3EtOH:1 xylene  1h

1EtOH:1 xylene  1h

1EtOH:3 xylene  1h

xylene                1h

xylene                1h

paraffin              4h

paraffin              4h

paraffin              4h