Table 1 Anticipated problems and suggested solutions in deploying the Rain-X® fixation method

Uneven Fixation, especially in larger animals

1. Pre-mix Rain-X® and FSW, then transfer animal to the mixture in the smallest volume possible or mix continuously during the addition of Rain-X®

Samples rupture or lyse on addition of paraformaldehyde or other secondary fixative

1. Extend Rain-X® treatment time before adding secondary fixative

Animals appear distorted

1. Relax animals by adding 7.5% MgCl before adding fixative, Add Rain-X® more quickly,

2. Pre-mix Rain-X® and FSW, then transfer animal to the mixture in the smallest volume possible

3. Reduce fixation time. As with any fixative, increased duration of fixation will eventually make the samples brittle and/or distorted.

Large animals appear distorted

1. Pre-mix Rain-X® and FSW, then transfer animal to the mixture in the smallest volume possible

Samples stick to plastic plates

1. Use glassware such as spot plates or scintillation vials

2. Use coated (“nonstick”) plasticware such as gelatin-coated plates or siliconized microfuge tubes

Samples do not fit into or get stuck inside transfer pipet

1. Cut tip to enlarge plastic pipet or pipet tip

2. Use polished glass, gelatin-coated Pasteur pipet

Sample initially appears well fixed but deteriorates during labeling

1. Perform washes in a larger volume

2. Use transfer pipette with wider mouth

3. Use less stringent detergent solution

Rain-X® droplets adhere to samples (usually appears as small, shiny “bubbles”)

1. Wash thoroughly after fixation

2. Add extra washes

Rain-X® droplets adhere to samples (not resolved by aqueous washes)

1. Dehydrate and rehydrate through graded alcohol series

Animals float to the top of the well and disintegrate at the air-water interface

1. Rain-X® will be disproportionally distributed across the well unless it is well mixed during the addition. Gently pipette up and down, or remove solution from the bottom of the well and gently add to the top to push the sample below the surface.

2. Try another vessel for fixation. We have found a glass spot plate to produce this effect less often than a multi-well plate.

Tissue starts to wrinkle during downstream applications

2. Keep the animals in a greater volume of water so that less is exposed to air