Jun 12, 2023

Public workspaceU54 SCENT Histology H&E Staining V.1

DOI
dx.doi.org/10.17504/protocols.io.4r3l22m9ql1y/v1
  • Shannon McCall1
  • 1Duke BioRepository & Precision Pathology Center, Duke University Hospital, Durham, NC, USA
Valerie Bekker
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.4r3l22m9ql1y/v1
Protocol Citation: Shannon McCall 2023. U54 SCENT Histology H&E Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22m9ql1y/v1Version created by Valerie Bekker
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 09, 2023
Last Modified: June 12, 2023
Protocol Integer ID: 83143
Abstract
To provide a standardized method of running, cleaning, refilling, and verifying quality of the automatic H&E stainer. Every morning before running slides for projects, a quality control slide containing brain, kidney, liver, and pancreas tissue is run, reviewed, and deemed acceptable by a histotechnician, who documents this review.
Materials
Personal Protective Equipment:
Lab coat
Gloves

Equipment:
Leica Autostainer XL
Microscope

Materials:
Bleach
Xylene
Alcohol, 100%
Alcohol, 95%
Alcohol, 80%
Hematoxylin –Pureview Hematoxylin 660+
Eosin – Pureview Eosin 615
Clearing Solution –Pureview Focus-AQ
Bluing – Pureview PH Blue
Chemical waste container
Wypall pads
Slide staining rack
Pre-cut H&E control slide
Protocol - Operating Autostainer and Running Daily Quality Control
Protocol - Operating Autostainer and Running Daily Quality Control
Open cover of autostainer and remove each lid of the reagents inside. Stack lids and set to the
right side of autostainer. Close cover.
Hit switch on the right side of autostainer to turn on and initialize.
Once initialization is complete, press button on screen to select staining program 1.
Take one slide from the box of unstained H&E control slides and label with:
a. H&E QA
b. Date
c. Tech initials
Place the slide into a black 30-count slide rack.
Pull the lever below the loading drawer on the autostainer to open.
Place slide rack inside and close.
Hit button to the left of loading drawer to begin staining run.
When staining has finished, autostainer will periodically beep.
Pull the lever below the exit drawer on the autostainer to open.
Press button next to exit drawer so instrument knows the rack was removed. (Autostainer will
continue to beep otherwise.)
Coverslip and microscopically verify correct staining of control tissues.
Denote onRHL Internal H&E Histologic Quality Check. If staining quality is suboptimal,
troubleshoot.
If staining is acceptable,write “PASS” on slide andstore in labeled control slide box next to
autostainer.
File form in H&E quality control binder at the end of the month.
Any research slides for the day can be loaded in the same way once the review is complete.

At the end of the workday, open cover and replace reagent lids to prevent evaporation. Close
cover.
Autostainer Program 1


Station #SolutionTime
1Xylene2 min
2Xylene2 min
3Xylene2 min
4100% Alcohol2 min
5100% Alcohol30 sec
695% Alcohol2 min*
795% Alcohol1 min
Wash 5Water wash1 min
8Hematoxylin*30 sec*
Wash 4Water Wash30 sec
Wash 3Water Wash2 min
9Clearing Solution30 sec
Wash 2Water Wash1 min
10Bluing Solution30 sec
Wash 1Water Wash30 sec
1180% Alcohol30 sec
12Eosin*30 sec
1395% Alcohol30 sec
1495% Alcohol30 sec
15100% Alcohol30 sec
16100% Alcohol30 sec
17Xylene30 sec
18Xylene30 sec
Exit (19)XyleneHold-exit station
*These are the only two stations with hard times. Slides may be held longer in other stations when multiple racks are being run at the same time.
Protocol - Changing the Autostainer
Protocol - Changing the Autostainer
At the end of the work week, remove all staining containers and empty into the chemical waste containers located below autostainer
Rinse containers out with water and, working over the sink, fill with a 10% bleach solution for at least 15 minutes or until visibly clean.
Rinse out containers and place upright on a counter that has been covered with Wypall pads. Leave to air dry over the weekend.
Fill water wash containers with 10% bleach and then 3 changes of waterto remove bleach residue (containers will automatically drain when filled).
Flush 10% bleach solution followed with 3 changes of water through the base of the stainer. Use a bleach-soaked Wypall pad to spot clean any splashed staining reagents. NOTE: Manual H&E stain line should remain set up until autostainer is refilled and tested the next week to ensure one staining line is always available for use.
At the beginning of the work week, refill staining containers and place back into autostainer.
Reagent setup is as follows:


ABCDEFGHIJKL
XyleneXyleneXylene100% Alcohol100% Alcohol95% Alcohol95% AlcoholWash 1Wash 2Wash 3Wash 4Wash 5
XyleneXyleneXylene100% Alcohol100% Alcohol95% Alcohol95% AlcoholEosin80% AlcoholBluing SolutionClearing SolutionHarris Hematoxylin

Mark reagents changed on Autostainer Maintenance Log.