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Dehydration / Rehydration in Immunostaining

Some histology preparations rely on removal of water from sections to aid preservation.

Most commonly rehydration is required to prepare Formalin Fixed Paraffin Embedded (FFPE) sections for staining and dehydration at the end of protocols where the samples are to be mounted in organic solvents to preserve the staining. Substrates such as DAB+Nickel and Vector's VIP must be dehydrated and mounted in non-aqueous media, others can be aqueously mounted, so it's important to check the conditions required. 

The timings below are a rough guide, for 3-5um sections. Thicker sections or whole mounts will need longer in each solution. 

Section Rehydration

1) Xylene or Xylene Substitute*   3-5 min to remove paraffin wax  

2)  Xylene or Xylene Substitute  3-5 min

3)  Xylene or Xylene Substitute  3-5 min

4) 100% ethanol         15sec.-3 min.

5) 100% ethanol         15sec.-3 min.

6) 95% ethanol           15sec.-3 min.

7) 95% ethanol           15sec.-3 min.

8) Water                      15sec.-3 min.

Dehydration & Clearing Steps after Immunostaining

Required for most non-aqueous mounting media, although VectaMount Express is a notable exception.  

1) 95% ethanol           15sec.-3 min.

2) 95% ethanol           15sec.-3 min. 

3) 100% ethanol         15sec.-3 min.

4) 100% ethanol         15sec.-3 min.

Clearing: (Removes water from paraffin section and usually requires just 3 min.  For previously frozen sections, clearing defats sections and may require up to 5 minute immersions)

5) Xylene or Xylene Substitute* 3-5 min.

6) Xylene or Xylene Substitute 3-5 min.

7) Xylene or Xylene Substitute 3-5 min.

8) Mount in non-aqueous/ permanent mounting media 

* Choice depends on stain compatibility and regulations for hazardous waste disposal.

Shop: Ethanol                                 Shop: non-aqueous mounting media

Industrial methylated spirits (IMS) are sometimes used in place of ethanol, as they are often cheaper, however, they are known to affect some enzyme substrates, so it is advisable to assess results before swapping. 

Incomplete removal of organic solvents / paraffin wax can prevent staining and detection reagents accessing the binding sites. 

Sections not dehydrated thoroughly before mounting in organic solvents can appear opaque and have water droplet artifacts. 

Be mindful of solution carry over; the most efficient way of handling slides is by using a metal slide rack to immerse the slides and there is usually some solution transferred between the immersion vessels with the slide rack. Over time this affects the relative percentages in each vessel, so a busy laboratory should have a regular schedule of replacing these solutions.  

Hydrophobic barriers may not be cleared by all xylene substitutes.