Jun 07, 2024

Public workspaceFast Green/Sirius Red Protocol For Leica ST5020 Automated Stainer

 Forked from Hematoxylin & Eosin Protocol For Leica ST5020 Automated Stainer
DOI
dx.doi.org/10.17504/protocols.io.ewov19djklr2/v1
Fast Green/Sirius Red Protocol For Leica ST5020 Automated Stainer
  • 1La Jolla Institute for Immunology
Zbigniew Mikulski
Open access
DOI: dx.doi.org/10.17504/protocols.io.ewov19djklr2/v1
Protocol Citation: Angela Denn, Zbigniew Mikulski 2024. Fast Green/Sirius Red Protocol For Leica ST5020 Automated Stainer. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19djklr2/v1
Manuscript citation:

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: December 02, 2021
Last Modified: June 07, 2024
Protocol Integer ID: 101393
Keywords: fibrosis, collagen, sirius red, picrosirius, picosirius, fast green, staining, st5020, automated staining, histology, special stain
Abstract
This Sirius Red Fast Green 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 thebest and most reproducible results are easier to achieve with an automated stainer.

This fork describes modifications to obtain high-quality special stain demonstrating collagen in FFPE and frozen tissues.
Image Attribution
La Jolla Institute for Immunology
Guidelines
Daily Maintenance for ST5020
A
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%RA) into aqueous waste.
Move reagent cup 5 into the 4 position.
Fill a clean, dry reagent cup with fresh (100%RA) and place in reagent cup 5 position.
Dispose of reagent cup 6 into aqueous waste and replace with fresh 90% RA.
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.
Dispose of reagent cup 31 (70% RA) into aqueous waste and replace with fresh 70% RA.
Dispose of reagent cup 29 (90% RA) into aqueous waste and replace with fresh 90% RA.
Dispose of reagent cups 16 and 17 (100% RA) 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% RA 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 (Propar) into aqueous waste.
Move reagent cup 14 (Pro-par) into the 26 position.
Fill a clean, dry reagent cup with fresh Pro-par and place into the 14 position.
Dispose of reagent cup 13 (Xylene) into aqueous waste.
Fill a clean, dry reagent cup with fresh xylene and place into the 13 position.
Record maintenance performed on the maintenance sheet.



Weekly Maintenance for ST5020

A
Remove cups 7, 8, 9, 10, 11, and 12.
Hand wash in hot soapy water using a sponge and 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

A
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.
Pour 1000ml of 10% bleach solution down drain.
Pour 3000ml of hot tap water down drain.
Calibrate touch screen.
Record maintenance performed on the maintenance sheet.

Quarterly Maintenance for ST5020
Replace active carbon filter quarterly


Materials
MATERIALS
ReagentPro-Par ClearantAnatech LTPCatalog #510
ReagentDirect-Red 80Merck MilliporeSigma (Sigma-Aldrich)Catalog #365548 ReagentReagent alcohol, 100%Pacific Southwest Lab EquipmentCatalog #374B
ReagentXyleneMedical Chemical CorporationCatalog #134B-1gl
ReagentFast Green FCFMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7252
ReagentSaturated Picric AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #P6744
ReagentGlacial acetic acidFisher ScientificCatalog #A38-500
ReagentDistilled WaterContributed by users

Solutions and Reagents:
  1. Fast Green solution
  2. Fast Green/Sirius Red solution
  3. Acidified water
  4. 90%, and 100% Reagent Alcohol
  5. Pro-Par Clearant

Prepare Fast Green solution:
DI water 500 ml
Fast Green 0.2 g

Prepare Fast Green/Sirius Red solution:
Saturated picric acid 500 ml
Fast Green 0.2 g
Direct Red 80 0.5 g

Acidified Water:
DI water 1000 ml
Glacial acetic acid 10 ml


Equipment
ST5020
NAME
Automated Multistainer
TYPE
Leica
BRAND
Leica ST5020
SKU
https://www.leicabiosystems.com
LINK
ST5020


Fast Green/Sirius Red Stain
Fast Green/Sirius Red Stain
Perform all required maintenance on the stainer and prepare working solutions
Arrange the reagents in the stainer according to the layout (note dark blue positions for FG/SR cups)

Reagent map for ST5020
Reagent map for ST5020



Program the FG/SR protocol into the ST5020 stainer according to the table below

Steps for staining
Steps for staining


Expected results
Expected results
Collagen fibers should be stained red and all other non-collagen proteins should be stained green.

There is strong positive staining in collagen fibers along blood vessels and minimal red staining in other tissue areas. Normal mouse liver sample scanned with 40x 0.95NA objective with Zeiss AxioScan Z1
There is strong positive staining in collagen fibers along blood vessels and minimal red staining in other tissue areas. Normal mouse liver sample scanned with 40x 0.95NA objective with Zeiss AxioScan Z1

In this mouse tumor sample there are abundant collagen fibers stained red. Minimal background staining in other, non-collagenous areas of the tissue. Sample scanned with 40x 0.95NA objective with Zeiss AxioScan Z1
In this mouse tumor sample there are abundant collagen fibers stained red. Minimal background staining in other, non-collagenous areas of the tissue. Sample scanned with 40x 0.95NA objective with Zeiss AxioScan Z1

Protocol references
Segnani C, Ippolito C, Antonioli L, Pellegrini C, Blandizzi C, Dolfi A, Bernardini N. Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon. PLoS One. 2015 Dec 16;10(12):e0144630. doi: 10.1371/journal.pone.0144630. PMID: 26673752; PMCID: PMC4682672.
https://pubmed.ncbi.nlm.nih.gov/26673752/