TITLE: UF / HuBMAP - H&E Staining Process 
AUTHORS: Franchesca Farris, Marda Jorgensen, Leigh Propper

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will detail our method of utilizing the H&E stain as a nonspecific entity to achieve optimal results in our staining process for our research samples.</div><div class = "text-block">Hematoxylin and Eosin stains are used in many areas of the laboratory, including frozen sections, fine needle aspirates, and paraffin fixed embedded tissues. There are multiple factors involved in determining what makes a well stained slide, and one of the primary factors involved in the final product is pathologist preference.  Other important factors also play important roles in your final stain, such as reagents and their quality, consistency, initial microtomy quality and selecting a sound procedure. </div><div class = "text-block">Hematoxylin is used to illustrate nuclear detail in cells. The resulting intensity and shade of the dye in the cells is primarily reliant on the length of time the sample spends in hematoxylin. The cytoplasmic component of the section is brought out by Eosin, which is the most commonly used counterstain in histology. It's varying shades of pink and red provide an exceptional contrast between the nuclear and cytoplasmic areas of interest. </div></div>

[STEPS]
?. Ensure your staining line or staining set up has sufficient reagents and is correctly set up for use.
?. Immerse slide holder in the first xylene staining pot for 5 minutes.
?. Remove slide holder from xylene and transfer to the first staining pot of 100% EtOH for 5 minutes.
Be sure to blot excess xylene before going into ethanol.
?. Transfer to 95% EtOH for 3 minutes.
?. Transfer to 70% EtOH for 3 minutes.
?. Transfer to 50% EtOH for 3 minutes.
?. Transfer to water for 2 minutes.
While sections are in water, skim surface of hematoxylin with a Kimwipe to remove oxidized particles.
?. Transfer slides to Hematoxylin for 1 minute and 15 seconds.
?. Rinse in de-ionized water,  and then transfer slides to tap water for 30 seconds.
?. After rinsing in tap water, place slide rack back into the first dish of water for 1 minute.
?. Transfer to Aqueous Clarifier for 45 seconds.
?. Do a quick rinse in de-ionized water and then transfer to tap water for 1 minute.
?. Transfer to bluing reagent for 1 minute.
?. Place in water for 5 minutes.
?. Transfer to 80% EtOH for 1 minute.
?. Transfer to Eosin-Y for 1 minute.
?. Transfer to 100% EtOH for 2 minutes.
?. Transfer to next xylene pot for 5 minutes.
?. After the second xylene change, use a xylene compatible mountant to affix a coverslip to the slide.
?. Immerse slide holder in the second xylene staining pot for 5 minutes.
?. Place slides into a xylene compatible slide rack.
?. Remove slide rack from first EtOH dish, and transfer to the second EtOH pot for an additional five minutes.
?. Transfer to the second change of water for an additional 2 minutes.
Blot excess water from slide holder before going into hematoxylin.
?. Transfer to second water staining dish for 1 minute.
?. Transfer to the second 100% EtOH for 5 minutes.
Be sure to blot excess ethanol before going into xylene.
?. Transfer to xylene for 5 minutes.