Aug 09, 2020
Version 1
Hematoxylin & Eosin Protocol For Leica ST5020 Automated Stainer V.1
- 1La Jolla Institute for Immunology
- La Jolla Institute Microscopy and Histology Core
Protocol Citation: Angela Denn 2020. Hematoxylin & Eosin Protocol For Leica ST5020 Automated Stainer. protocols.io https://dx.doi.org/10.17504/protocols.io.bf2ejqbe
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: May 05, 2020
Last Modified: June 07, 2024
Protocol Integer ID: 36646
Keywords: hematoxylin, eosin, h&e
Abstract
This Hematoxylin and Eosin protocol is for the Leica ST5020 Automated multistainer. You can use it as a guide to developing your own protocol, catered to the needs of your researchers. This template can be modified for manual staining but best and most reproducible results are easier to achieve with an automated stainer.
Guidelines
Daily Maintenance for ST5020
| Filter reagent cup 21 (Hematoxylin 560) using a funnel, Whatman #1 filter paper, and 500ml Erlenmeyer flask | |
| Dispose of reagent cup 1 (Pro-par) into aqueous waste | |
| Move reagent cup 2 into the 1 position | |
| Move reagent cup 3 into the 2 position | |
| Fill a clean, dry reagent cup with fresh Pro-par and place in reagent cup 3 position | |
| Dispose of reagent cup 4 (100%IPA) into aqueous waste | |
| Move reagent cup 5 into the 4 position | |
| Fill a clean, dry reagent cup with fresh (100%IPA) and place in reagent cup 5 position | |
| Dispose of reagent cup 6 into aqueous waste and replace with fresh 90%IPA | |
| Dispose of reagent cup 33 (Define) into aqueous waste and replace with fresh Define | |
| Dispose of reagent cup 32 (Blue Buffer 8) into aqueous waste and replace with fresh Blue Buffer 8 in a clean, dry reagent cup | |
| Dispose of reagent cup 31 (70% IPA) into aqueous waste and replace with fresh 70% IPA | |
| Dispose of reagent cup 29 (90% IPA) into aqueous waste and replace with fresh 90% IPA | |
| Dispose of reagent cups 16 and 17 (100% IPA) into aqueous waste | |
| Move reagent cup 15 into the 17 position | |
| Move reagent cup 28 into the 16 position | |
| Move reagent cup 27 into the 15 position | |
| Fill clean, dry reagent cups with fresh 100% IPA and place into the 27 and 28 position | |
| Add approximately 20 grams of DriRite to reagent cups 27 and 28 | |
| Dispose of reagent cup 26 (50:50 Propar/100% IPA) into aqueous waste | |
| Fill clean, dry reagent cup with 50:50 Propar/100% IPA. Add ~20 grams of DriRite and place in position 26 | |
| Dispose of reagent cup 14 (Pro-par) into aqueous waste | |
| Move reagent cup 13 into the 14 position and add ~20 grams of Dri-Rite | |
| Move reagent cup 25 into the 13 position | |
| Fill clean, dry reagent cup with fresh Pro-par and place in position 25 | |
| Record maintenance performed on the maintenance sheet |
Weekly Maintenance for ST5020
| Remove cups 7, 8, 9, 10, 11, and 12 | |
| Hand wash in hot soapy water using a sponge and Dawn dish detergent | |
| Rinse thoroughly in hot water | |
| Soak cups in a 10% bleach solution for ten minutes | |
| Rinse thoroughly in hot water | |
| Replace cups into position | |
| Record maintenance performed onto the maintenance sheet |
Monthly Maintenance for ST5020
| Dispose of reagent cup 21 (Hematoxylin 560) into aqueous waste | |
| Fill clean, dry reagent cup with fresh Hematoxylin 560 and place into the 21 position | |
| Dispose of reagent cup 18 (Eosin Phloxine 515) into aqueous waste | |
| Fill clean, dry reagent cup with fresh Eosin Phloxine 515 and place into the 18 position | |
| Record maintenance performed on the maintenance sheet |
Materials
MATERIALS
Pro-Par ClearantAnatech LTPCatalog #510
Isopropyl Alcohol 99%Pacific Southwest Lab EquipmentCatalog #104B
SelecTech HematoxylinLeica BiosystemsCatalog #3801571
SelecTech Eosin 515 PhloxineLeica BiosystemsCatalog #3801606
SelecTech DefineLeica BiosystemsCatalog #3803591
SelecTech Blue Buffer 8Leica BiosystemsCatalog #3802916
Solutions and Reagents:
- Leica SelecTech Hematoxylin
- Leica SelecTech Eosin 515 Phloxine
- Leica SelecTech Define
- Leica SelecTech Blue Buffer 8
- Isopropyl Alcohol, 99%
- Pro-Par Clearant
Prepare working Define solution by mixing 2 capfuls of Define filled to the fill line with 500 mls of deionized water.
Prepare working Blue Buffer 8 solution by mixing 2 capfuls of Blue Buffer 8 filled to the fill line with 500 mls of deionized water.
Hematoxylin and Eosin solutions are ready to use.
Equipment
ST5020
NAME
Automated Multistainer
TYPE
Leica
BRAND
Leica ST5020
SKU
LINK
H&E Stain
H&E Stain
Reagent layout for multistain function
| STEP | STATION | DYE/REAGENT STATION | DURATION | EXACT | DIP | |
| 1 | 38 | Oven station | 5:00 | no | no | |
| 2 | 37 | Oven station | 5:00 | no | no | |
| 3 | 1 | Pro-Par | 5:00 | no | yes | |
| 4 | 2 | Pro-Par | 5:00 | no | yes | |
| 5 | 3 | Pro-Par | 5:00 | no | yes | |
| 6 | 4 | 100% IPA | 1:30 | no | yes | |
| 7 | 5 | 100% IPA | 1:30 | no | yes | |
| 8 | 6 | 90% IPA | 1:30 | no | yes | |
| 9 | 12 | DI Water Wash | 1:30 | no | no | |
| 10 | 21 | SelectTech Hematoxylin 560 | 1:30 | yes | yes | |
| 11 | 11 | DI Water Wash | 1:30 | no | no | |
| 12 | 33 | SelectTech Define | 1:00 | yes | no | |
| 13 | 10 | DI Water Wash | 2:00 | no | no | |
| 14 | 32 | SelectTech Blue Buffer 8 | 1:30 | yes | no | |
| 15 | 9 | DI Water Wash | 1:30 | no | no | |
| 16 | 31 | 70% IPA | 1:00 | no | yes | |
| 17 | 18 | ST Alcoholic Eosin Y 515 | 0:10 | yes | yes | |
| 18 | 29 | 90% IPA | 0:20 | yes | yes | |
| 19 | 17 | 100% IPA | 0:30 | yes | yes | |
| 20 | 16 | 100% IPA | 1:00 | no | yes | |
| 21 | 15 | 100% IPA | 1:30 | no | yes | |
| 22 | 28 | *100% IPA | 1:30 | no | yes | |
| 23 | 27 | *100% IPA | 1:30 | no | yes | |
| 24 | 26 | *50:50 Pro-par/100% IPA | 1:00 | yes | yes | |
| 25 | 14 | *Pro-Par | 5:00 | no | yes | |
| 26 | 13 | Pro-Par | 5:00 | no | yes | |
| 27 | EXIT | Pro-Par |