Mar 20, 2025
Haematoxylin and Eosin (H&E) staining of paraffin sections
- Samson Dowland1
- 1Charles Perkins Centre Histology Facility, The University of Sydney, NSW, 2006, Australia
- The University of Sydney
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 17, 2025
Last Modified: March 20, 2025
Protocol Integer ID: 124459
Keywords: Histology, Staining, Microscopy, Pathology, Haematoxylin, Eosin, initial red colour of the haematoxylin, haematoxylin dye, rich contrast to the other tissue component, histological staining technique, used histological staining technique, paraffin sections haematoxylin, staining biological sample, stained cytoplasm, alum haematoxylin, rich in proteoglycan, erythrocyte, nuclear staining, other tissue component, haematoxylin, clear overview of cellular structure, proteoglycan, tissue for diagnostic purpose, cytoplasm of most other cell type, other cell type, differentiation between cytoplasm, most other cell type, tissue architecture, routine histopathological examination, cellular structure, cell, cytoplasm, standard choice for routine histopathological examination, biological samples for biomedical research, eosin, connective tissue, tissue, bluing solution, areas of bone, cartilage, initial red colour, staining of paraffin sections haematoxylin, staining, alkaline ph of scott
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
Hydrochloric AcidBanksia Scientific Company (AJAX FineChem)Catalog #AJA256
EthanolPOCD ScientificCatalog #ETHABS70WV5
Eosin Alcoholic 0.1% with Phloxine (SNP Formula)POCD ScientificCatalog #SNPEOSIN5L
EthanolHurst ScientificCatalog #EIMS95-5L
EthanolPOCD ScientificCatalog #ETHABS20M
XylenePOCD ScientificCatalog #XYL5P
DPX Mountant for histologyMerck MilliporeSigma (Sigma-Aldrich)Catalog #06522
Harris HaematoxylinPOCD ScientificCatalog #HHXIMP
Sodium hydrogen carbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #401676
Magnesium sulphateChemSupplyAustraliaCatalog #ML073
Troubleshooting
Deparaffinisation and rehydration
32m
Nuclear Staining
5m 30s
Stain in Harris's Haematoxylin
Harris HaematoxylinPOCD ScientificCatalog #HHXIMP
2m
Wash with running tap water to remove excess stain
2m
Differentiate in acid alcohol (5 - 10 dips)
[1% Hydrochloric Acid (HCl) in 70% Ethanol]
Hydrochloric AcidBanksia Scientific Company (AJAX FineChem)Catalog #AJA256
EthanolPOCD ScientificCatalog #ETHABS70WV5
Wash in tap water
30s
Blue in Scott's Bluing Solution
[Sodium hydrogen carbonate 2.5g/L; Magnesium sulphate 20g/L; in distilled water]
Sodium hydrogen carbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #401676
Magnesium sulphateChemSupplyAustraliaCatalog #ML073
30s
Wash in tap water
30s
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.
Counterstaining
1m 20s
70% Ethanol
EthanolPOCD ScientificCatalog #ETHABS70WV5
Counterstain in Eosin - 2 changes, 40 seconds each
Eosin Alcoholic 0.1% with Phloxine (SNP Formula)POCD ScientificCatalog #SNPEOSIN5L
1m 20s
Dehydration, clearing and mounting
16m
95% Ethanol
EthanolHurst ScientificCatalog #EIMS95-5L
1m
95% Ethanol
EthanolHurst ScientificCatalog #EIMS95-5L
1m
100% Ethanol
EthanolPOCD 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
EthanolPOCD 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
XylenePOCD ScientificCatalog #XYL5P
5m
100% Xylene
XylenePOCD ScientificCatalog #XYL5P
5m
Coverslip with a resinous mounting medium, such as DPX
DPX Mountant for histologyMerck MilliporeSigma (Sigma-Aldrich)Catalog #06522
Results
54m 50s
Expected result
Nuclei: blue-purple
Cytoplasm: pink
Erythrocytes: pink-red
Collagen: pink
Muscle: pink-red
Cartilage: pink-blue
Bone: pink-purple
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.