Different progressive hematoxylin stains for histological examination of myocardium, blood vessels, liver and spleen

Aim of the study was to compare the distinct types of progressive hematoxylin stains and to optimize the protocols of hematoxylin and eosin staining of blood vessels, heart, liver and spleen.

Material and methods. Heart (ventricles), abdominal aorta, liver (right lobe) and spleen (left part) of the Wistar rats were excised, fixed in 10% neutral phosphate buffered formalin for 24 h, washed in tap water for 2 h, dehydrated in ascending ethanol series (70 % 80 %, and 95 %) and isopropanol, embedded into paraffin and then sectioned (5 pm) using rotary microtome. Staining was performed using Mayer’s, Gill’s, or Carazzi’s hematoxylin during 2, 5, or 15 minutes and 1 % alcoholic/aqueous eosin for 2 minutes without differentiative solution. Results were assessed by three independent histologists.

Results. All examined progressive hematoxylin stains had their distinctive features. Mayer’s hematoxylin demonstrated the most intensive nuclear staining; however, staining for 15 minutes could lead to the bluing of cytoplasm and extracellular matrix. In contrast, Gill’s hematoxylin was characterized by less intensive nuclear staining and achieved clear blue-violet shade only after 15 minutes of staining. Carazzi’s hematoxylin showed balanced coloration of nuclei and cytoplasm/ extracellular matrix and did not change the red/pink shades of eosin, yet the intensity of nuclear staining was less as compared to Mayer’s hematoxylin. Short-term (2 minutes) staining was insufficient to reach intensive nuclear staining.

Conclusion. The optimal hematoxylin and eosin staining protocol is to use eosin for 2 minutes following staining by Carazzi’s hematoxylin for 15 minutes (for aorta), Carazzi’s or Gill’s hematoxylin for 15 minutes or Mayer’s hematoxylin for 5 minutes (for liver), Carazzi’s or Gill’s hematoxylin for 15 minutes (for heart), and Carazzi’s hematoxylin for 5 minutes (for spleen).

1. Меркулов Г. А. Курс патологогистологической техники. 5-е изд., испр. и доп. Л.: Медицина, 1969. 423 с.

2. Коржевский Д.Э. Применение гематоксилина в гистологической технике. Морфология. 2007; 132 (6): 77-81.

3. Коржевский Д.Э., Гилерович Е.Г., Кирик О.В., Сухорукова Е.Г., Григорьев И.П. Морфологическая диагностика: подготовка материала для морфологического исследования и электронной микроскопии. СПб.: СпецЛит, 2013. С. 47-58.

4. Prokopyev Yu.A., Fomenko D.V., Kizichenko N.V., Kudryavtseva Yu.A., Borisov V.V., Popova A.P., Mikhaylova N.N. Methodical approaches to study of the anthracosilicosis as a factor of risk to morphological deseases of blood vessels. Kompleksnye prob-lemy serdechno-sosudistykh zabolevaniy = Complex Issues of Cardiovascular Diseases. 2015; 4 (3): 23-27. [In Russian].

5. Avwioro G. Histochemical uses of haematoxylin. JPCS. 2011; 1 (5): 24-34.

6. Bancroft J.D., Layton C. The hematoxylins and eosin. In: Bancroft’s theory and practice of histological techniques, 8th ed. Eds. K. Suvarna, C. Layton, J. Bancroft. Elsevier, 2019: 126-139.

7. Chan J.K. The wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology. Int. J. Surg. Pathol. 2014; 22 (1): 12-32. doi: 10.1177/1066896913517939

8. Feldman A.T., Wolfe D. Tissue processing and hematoxylin and eosin staining. Methods Mol. Biol. 2014; 1180: 31-43. doi: 10.1007/978-1-4939-1050-2_3

9. Fischer A.H., Jacobson K.A., Rose J., Zeller R. Hematoxylin and eosin staining of tissue and cell sections. cSh Protoc. 2008; 2008: pdb.prot4986. doi: 10.1101/pdb.prot4986

10. Li Y., Li N., Yu X., Huang K., Zheng T., Cheng X., Zeng S., Li X. Hematoxylin and eosinstaining of intact tissues via delipidation and ultrasound. Sci. Rep. 2018; 8 (1): 12259. doi: 10.1038/s41598-018-30755-5

11. Martina J.D., Simmons C., Jukic D.M. Highdefinition hematoxylin and eosin staining in a transition to digital pathology. J. Pathol. Inform. 2011; 2: 45. doi: 10.4103/2153-3539.86284

12. Senturk G.E., Canillioglu Y.E. Which histochemical staining technique should I choose for biological specimens. In: Microscopy: advances in scientific research and education. Ed. A. Mendez-Vilas. Forma-tex Research Center, 2014: 769-775.

13. Titford M. The long history of hematoxylin. Biotech. Histochem. 2005; 80 (2): 73-78. doi: 10.1080/10520290500138372

14. Turkki R., Linder N., Kovanen P., Pellinen T., Lundin J. Identification of immune cell infiltration in hematoxylineosin stained breast cancer samples: texture-based classification of tissue morphologies. Proc. SPIE. 2016; 9791: 979110. doi: 10.1117/12.2217040

15. Zarella M.D., Yeoh C., Breen D.E., Garcia F.U. An alternative reference space for H&E color normalization. PLoS One. 2017; 12 (3): e0174489. doi: 10.1371/journal.pone.0174489