[Histonet] Ada's Mail Stain line order

Dee Wolfe deewolfe at anatechltdusa.com
Tue Oct 26 12:41:52 CDT 2021

Hi Anne,

Are they using the term "clearing" loosely, but really mean "rinsing"? The term "clearing" typically refers to the step after the alcohol (dehydration) and before coverslipping (if using a permanent, solvent based mounting media). Not sure why someone would "clear" before the rinse and bluing step in frozen section staining.

For paraffin embedded specimens, the slide would need to Deparaffinize (in clearing agent-xylene or xylene substitute), dehydrate (graded alcohols—high to low), water rinse (preferably DI), stain the nuclei (hematoxylin), water rinse, decolorize (acid alcohol-if staining regressively), water rinse(s), bluing, water rinses, graded alcohol–low to high, cytoplasmic staining (Eosin), alcohol rises, clearing.

For frozen section: 100% alcohol (or other fixative), water rise, hematoxylin (usually progressive, so no need to decolorize), bluing, water rinse, etc…

You may choose to skip bluing as tap water can do it. Although for consistency it should be kept in (tap water pH can with seasons, water treatment facilty processes, etc.)

Dee Wolfe

On Oct 26, 2021, at 12:26 PM, Anne Murvosh via Histonet wrote:

> When doing frozen section staining for Mohs we have DI water and don't need a clearing agent. I am helping set up someone who is using tap water and needs to clear it. They mentioned that they used to clear first, rinse, then use bluing. I thought it was the opposite after heme. Rinse, bluing, rinse, clearing. I don't remember which is first. Actually do you need a bluing if using tap water since that blues better then DI water? Thanks for your input, it's been 10 years since I had to think about this. Anne
> Anne Murvosh
> Histology Technician
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