801.1.HTC_H&E Stain (Paraffin or Cryosections)
Gloria S Pryhuber, Cory Poole, Anthony Corbett, Jeffrey Purkerson, Luis Colon
Abstract
Hematoxylin and Eosin staining is the standard chemical stain used on slides to be reviewed for assessment of general histopathology and the generation of a pathology report for each donor.
(From Wikipedia) H&E is the combination of two histological stains: hematoxylin and eosin. The hematoxylin stains cell nuclei a purplish blue, and eosin stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors. Hence a pathologist can easily differentiate between the nuclear and cytoplasmic parts of a cell, and additionally, the overall patterns of coloration from the stain show the general layout and distribution of cells and provides a general overview of a tissue sample's structure. Pattern recognition, by expert humans or by software that aids those experts in digital pathology, provides histologic information.
This stain combination was first introduced in 1876 by A. Wissowzky.
This protocol version accomodates removal of Phenocycler flow cell prior to H&E staining.
Before start
Prepare the bluing solution by adding 1 mL of 28% Sodium Hydroxide to 400 mL of water1. The Hematoxylin may need to be gravity filtered through the Whatman filter paper before use
- The Eosin/Phloxine is in a ready-to-use state
Steps
Staining Procedure
Verify that all slides are completely dry. Slides can be dried in an 55C oven for one hour or more prior to use to prevent loss of tissue.
If sections were from tissue block embedded in paraffin, deparaffinize slides with 3 changes of Xylene for 5 minutes each.
If sections are from fresh frozen, unfixed tissue, consider a brief fixation with methanol or paraformaldyhyde prior to staining.
If sections are post Phenocycler-Fusion imaging and Flow cells have been removed by incubation of slides in Xylene for 2-4 h proceed to step 5.
Rehydrate slides by submerging them in Ethanol in the following order for 3 minutes each.
i. 100% Ethanol
ii. 100% Ethanol
iii. 95% Ethanol
iv. 70% Ethanol
v. Wash the slides briefly with 3 changes of water and leave them in the final water for 5 minutes
Place the slides in Hematoxylin for 1 minute. Adjust timing by 30 second intervals to lighten or darken the stain.
Wash slides off with water until the purple color is no longer coming off into the water. Leave them in the final water for 5 minutes.
Briefly dip the slides in the bluing solution (1 ml of 28% Ammonium Hydroxide (NH4OH) in 400 ml distilled water, dilution good for ~1 month).
Wash the slides with 3 changes of water and leave in the final water for 5 minutes.
Dehydrate the slides in 95% Ethanol for 3 minutes
Place the slides in Eosin/Phloxine for 1-2 minutes
Wash the slides for 3 minutes each in the following solutions
i. 95% Ethanol,
ii. 95% Ethanol
iii. 100% Ethanol
iv. 100% Ethanol
Place the slides through 3 changes of Xylene (NOT the same Xylene used for deparaffinizing the slides) for 3-5 minutes each.
Coverslip the slides by removing them from the final xylene (1 at a time) and dabbing the edges with paper towel to absorb some excess Xylene. Do not allow the section to dry out. Place 1-2 drops of mounting media on the tissue section and carefully place the coverslip starting on at an angle and laying over the tissue to avoid air bubbles. Carefully use the cotton tipped applicator to push out any excess mounting media and air bubbles.
Place the slides, tissue side up, in a slide folder to dry overnight.
The next day the slides can be cleaned by using a cotton tipped applicator to apply Xylene to the front and back of the slide as needed to remove excess mounting media. A Kimwipe can be used to wipe off the applied Xylene and cleaning any residual debris from the slide, making sure to use a new section of the Kimwipe for each side.
Leave the slides overnight to dry further.
Best to place the slides in a slide box for longer term storage
Imaging H&E Stained Tissue
For HuBMAP, following Phenocycler MxF, cover-slipped H&E stained slides are imaged on the PhenoImager using the manufacturer's H&E protocol, 20X Objective, with resolution = 0.51 x 0.51 pixel size.
H&E Images are uploaded to local Omero web database after conversion from .qptiff format
Download Command Line Tools from https://www.openmicroscopy.org/bio-formats/downloads/ https://www.openmicroscopy.org/bio-formats/downloads/
Execute the following in command prompts:
C:>Set BF_MAX_MEM=512M
"C:\Program Files\bftools\bfconvert.bat" -series 0 -compression LZW -pyramid-resolutions 5 -pyramid-scale 2
Upload the resulting ome.tiff file to the local URMC CODEX folder in Omero and the respective tissue donor subfolder (e.g. D265) utilizing the Omero importer.
