Mar 20, 2025

Public workspaceHaematoxylin and Eosin (H&E) staining of paraffin sections

DOI
dx.doi.org/10.17504/protocols.io.n92ld5b3xv5b/v1
  • 1Charles Perkins Centre Histology Facility, The University of Sydney, NSW, 2006, Australia
  • The University of Sydney
Samson Dowland
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DOI: dx.doi.org/10.17504/protocols.io.n92ld5b3xv5b/v1
Protocol Citation: Samson Dowland 2025. Haematoxylin and Eosin (H&E) staining of paraffin sections. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld5b3xv5b/v1
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: March 16, 2025
Last Modified: March 20, 2025
Protocol Integer ID: 124459
Keywords: Histology, Staining, Microscopy, Pathology, Haematoxylin, Eosin
Abstract
Haematoxylin and Eosin (H&E) staining is the most-commonly used histological staining technique. This method provides a clear overview of cellular structure and tissue architecture, making it the standard choice for routine histopathological examination of tissue for diagnostic purposes and a popular option when staining biological samples for biomedical research or teaching purposes.

The Harris's Haematoxylin used here is an alum haematoxylin, meaning it uses an aluminium salt as a mordant. Once nuclear staining is complete, the alkaline pH of Scott's bluing solution is used to convert the initial red colour of the haematoxylin to a light blue. Areas of bone and cartilage that are rich in proteoglycans will also take up the haematoxylin dye and stain blue-purple. Subsequent counterstaining with the combined Eosin Y + Phloxine B solution provides rich contrast to the other tissue components, enhancing differentiation between cytoplasm, muscle and connective tissue. In particular, erythrocytes are stained vibrantly and appear almost red, making them stand out against the pink-stained cytoplasm of most other cell types.
Protocol materials
ReagentEthanolHurst ScientificCatalog #EIMS95-5L
In 2 steps
ReagentEthanolPOCD ScientificCatalog #ETHABS20M
In 2 steps
ReagentDPX Mountant for histologyMerck MilliporeSigma (Sigma-Aldrich)Catalog #06522
Step 17
ReagentSodium hydrogen carbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #401676
Step 6
ReagentMagnesium sulphateChemSupplyAustraliaCatalog #ML073
Step 6
ReagentEthanolPOCD ScientificCatalog #ETHABS70WV5
In 2 steps
ReagentEosin Alcoholic 0.1% with Phloxine (SNP Formula)POCD ScientificCatalog #SNPEOSIN5L
Step 10
ReagentXylenePOCD ScientificCatalog #XYL5P
In 2 steps
ReagentHarris HaematoxylinPOCD ScientificCatalog #HHXIMP
Step 2
ReagentHydrochloric AcidBanksia Scientific Company (AJAX FineChem)Catalog #AJA256
Step 4
Deparaffinisation and rehydration
Deparaffinisation and rehydration
32m
32m
32m
Nuclear Staining
Nuclear Staining
5m 30s
5m 30s
Stain in Harris's Haematoxylin

ReagentHarris HaematoxylinPOCD ScientificCatalog #HHXIMP

2m
Wash with running tap water to remove excess stain
2m
Wash
Differentiate in acid alcohol (5 - 10 dips)

[1% Hydrochloric Acid (HCl) in 70% Ethanol]
ReagentHydrochloric AcidBanksia Scientific Company (AJAX FineChem)Catalog #AJA256
ReagentEthanolPOCD ScientificCatalog #ETHABS70WV5

Critical
Wash in tap water
30s
Wash
Blue in Scott's Bluing Solution

[Sodium hydrogen carbonate 2.5g/L; Magnesium sulphate 20g/L; in distilled water]
ReagentSodium hydrogen carbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #401676
ReagentMagnesium sulphateChemSupplyAustraliaCatalog #ML073


30s
Wash in tap water
30s
Wash
Examine slides under microscope to assess nuclear staining. Nuclei should be blue, cytoplasm and collagen should be grey. If sections remain overstained (cytoplasm and other tissue components remain blue/purple) repeat steps 4-8. If sections have been over-differentiated, return to step 2.
Imaging
Counterstaining
Counterstaining
1m 20s
1m 20s
70% Ethanol

ReagentEthanolPOCD ScientificCatalog #ETHABS70WV5

Counterstain in Eosin - 2 changes, 40 seconds each

ReagentEosin Alcoholic 0.1% with Phloxine (SNP Formula)POCD ScientificCatalog #SNPEOSIN5L

1m 20s
Dehydration, clearing and mounting
Dehydration, clearing and mounting
16m
16m
95% Ethanol

ReagentEthanolHurst ScientificCatalog #EIMS95-5L

1m
95% Ethanol

ReagentEthanolHurst ScientificCatalog #EIMS95-5L

1m
100% Ethanol

ReagentEthanolPOCD ScientificCatalog #ETHABS20M
Note: ensure whole slide and slide rack is fully immersed in ethanol to remove all traces of water, preventing water contamination of the xylene
2m
100% Ethanol

ReagentEthanolPOCD ScientificCatalog #ETHABS20M
Note: ensure whole slide and slide rack is fully immersed in ethanol to remove all traces of water, preventing water contamination of the xylene
2m
100% Xylene

ReagentXylenePOCD ScientificCatalog #XYL5P

5m
100% Xylene

ReagentXylenePOCD ScientificCatalog #XYL5P

5m
Coverslip with a resinous mounting medium, such as DPX

ReagentDPX Mountant for histologyMerck MilliporeSigma (Sigma-Aldrich)Catalog #06522

Results
Results
54m 50s
54m 50s

Expected result
Nuclei: blue-purple
Cytoplasm: pink
Erythrocytes: pink-red
Collagen: pink
Muscle: pink-red
Cartilage: pink-blue
Bone: pink-purple


Mouse retina

Mouse oesophagus. Demonstrates staining in keratin, epithelium, connective tissue and muscle.


Mouse bronchus, demonstrating staining of cartilage.

Mouse skeletal muscle.





Protocol references
Kiernan, J. (2015). Histological and histochemical methods. Scion publishing ltd.
Avwioro, G. (2011). Histochemical uses of haematoxylin—a review. Jpcs, 1(5), 24-34.
https://www.stainsfile.com/theory/methods/hematoxylin-eosin-staining/eosin/