Aug 07, 2019

Public workspaceU Michigan - Sirius Red staining V.2

DOI
dx.doi.org/10.17504/protocols.io.56tg9en
  • Jeff Hodgin1
  • 1University of Michigan - Ann Arbor
  • Mouse Metabolic Phenotyping Centers
    Tech. support email: info@mmpc.org
Lili Liang
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DOI: dx.doi.org/10.17504/protocols.io.56tg9en
External link: https://mmpc.org/shared/document.aspx?id=313&docType=Protocol
Protocol Citation: Jeff Hodgin 2019. U Michigan - Sirius Red staining. protocols.io https://dx.doi.org/10.17504/protocols.io.56tg9en
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: August 05, 2019
Last Modified: August 07, 2019
Protocol Integer ID: 26547
Keywords: Sirius red staining, Tubulointerstitial disease
Abstract
Summary:

Picrosirius red staining (also called Sirius red staining) highlights fibrosis by staining collagen (collagen 1 and III fibers) fibers in paraffin-embedded kidney tissue sections. Sirius red stained collagen appears red by light microscopy. When polarized, the birefringence is highly specific for collagen. The larger collagen fibers are bright yellow or orange, and the thinner ones, including reticular fibers, are green.
Materials
MATERIALS
ReagentSirius Red (Direct Red 80)Merck MilliporeSigma (Sigma-Aldrich)Catalog #365548
ReagentPicric acidFisher ScientificCatalog #5860-1
ReagentAcidified Water
ReagentHematoxylin 2 Richard-Allan ScientificCatalog #7231
ReagentAcetic acid GlacialFisher ScientificCatalog #A38-212
ReagentXyleneFisher ScientificCatalog #X3P-1GAL
ReagentEthanol (EtOH) 200 ProofDecon LaboratoriesCatalog #2701
ReagentPer mountThermo ScientificCatalog #8312-4
ReagentCover slip Fisher ScientificCatalog #12-542-B
Reagent Preparation:

Reagent 1: Picro-Sirius Red Solution:
Reagents and Materials: Sirius Red (Direct Red 80) powder, saturated aqueous solution of picric acid

Procedure: Mix 0.5g Sirius red powder with 500 ml of saturated aqueous solution of picric acid. This solution is stable for at least 3 years and can be re-used

Reagent 2: Acidified Water
Reagents and Materials: Acetic acid and distilled water (milliQ water)

Procedure: Mix 5 ml of acetic acid with 1000 ml water.


Note:
Sigma-Aldrich, RRID:SCR_008988
Fisher Scientific, RRID:SCR_008452
Wash 2x for 5 min in Xylene
Wash 2x for 3 min in 100% EtOH
Wash 1x for 2 min in 95% EtOH
Wash 1x for 2 min in 80% EtOH
Wash 1x for 1 min in 70% EtOH
Wash in running tap water for 3 min
Incubate in hematoxylin for 8 min.
Wash in running tap water for 10 min
Incubate for 60 min in Picrosirius red solution (do not shorten this time)
Wash 2x for 5 min in acidified H²O (Change after each wash)
Wash 1x for 1 min in 70% EtOH
Wash 1x for 1 min in 80% EtOH
Wash 1x for 1 min in 95% EtOH
Wash 1x for 1 min in 100% EtOH
Wash 2x for 3 min in Xylene
Mount slides with mounting medium - 1 drop
Insert cover glass carefully, avoid bubble