**9. Materials and methods for histology sections**

Samples of the skin tissue of different reptile species were preserved in 5% formaldehyde. Pieces of different types of tissue were included in the paraffin by using usual procedure with the apparatus Leica TP1020. Histological slides were prepared before use by the procedure which ensured that histological sections adhered to the slides properly and prevented sections from falling off the slides during further procedures. Tissue samples embedded in paraffin were cut by a hand microtome (Leica) into 5-μm-thick slices, which were transferred with brushes onto the smooth surface of warm water bath (40°C) and from there on the microscopic slides. Histological slides were dried in a thermostat (50°C). For classical histological staining with hematoxylin-eosin (H&E), samples were deparaffinized in xylene substitute (Neoclear; Merck) (2 × 5 minutes) and afterward rehydrated in decreasing concentrations of ethyl alcohol (100% for 2 × 5 minutes, 96% for 5 minutes, 75% for 5 minutes) and distilled water (2 × 5 minutes). In the following steps, samples were stained with either hematoxylin (Merck) (2 minutes), washed in running water (20 minutes), stained with eosin (1–2 minutes), and washed in distilled water (5 minutes) or Toluidine blue dye solution (20 minutes) and washed in distilled water (5 minutes) three times. Further, dehydration in ethyl alcohol with increasing concentration was performed (75% for approximately 5 minutes [depending on the sample; appropriate timing is observed during staining for intensity of reaction], 96% for 1× around 5 minutes, 100% for 2 × 5 minutes). Clarification of samples after drying and staining was carried out in xylene substitute (Neoclear) (3 × 5 minutes). At the end, a drop of Neo-Mount medium (Merck) was applied onto each tissue sample and the sample was covered with cover slide. Pictures were taken on Nikon FXA microscope with Nikon DS-F1 camera and transferred to program for image analysis by Lucia-G.

## **Acknowledgements**

Authors would like to acknowledge financial support from ARRS, Research group P4-0053; Jasna Šporar for technical assistance with histological samples;

**153**

**Author details**

proof-reading.

Catrin Sian Rutland1

Leicestershire, UK

Ljubljana, Slovenia

Medicine Vienna, Austria

provided the original work is properly cited.

, Pia Cigler2

\*Address all correspondence to: valentina.kubale@vf.uni-lj.si

*Reptilian Skin and Its Special Histological Structures DOI: http://dx.doi.org/10.5772/intechopen.84212*

Prof. Alenka Dovč and Zlatko Golob, PhD, for their help with providing diverse reptile samples; and Catrin S. Rutland for winning the Women in Science award, which resulted in us preparing this chapter and book, and for English language

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Valentina Kubale3

1 School of Veterinary Medicine and Science, University of Nottingham,

2 Small Animal Clinic, Service for Birds and Reptiles, University of Veterinary

3 Veterinary Faculty, Institute for Preclinical Sciences, University of Ljubljana,

\*

*Reptilian Skin and Its Special Histological Structures DOI: http://dx.doi.org/10.5772/intechopen.84212*

*Veterinary Anatomy and Physiology*

**dermatology of reptiles**

promotes better healing and strength.

able and more vulnerable to parasites and infection.

**9. Materials and methods for histology sections**

off at irregular intervals and it takes longer periods.

2 weeks [8, 32]. In turtles and crocodiles, sloughing of the skin arises to a lesser extent and it is comparable to that of birds and mammals, in whom small flakes fall

**8. Clinical importance of histology and anatomy knowledge for the** 

Reptile skin heals much slower than mammalian skin, often taking about 6 weeks to fully restore the defect. Malnourished animals are hypoproteinemic and unable to produce enough enzymes to form true cleavage zone, resulting in dysecdysis (failure to shed). Lack of moisture will also delay the process [11]. Skin permeability increases when skin is in contact with water, so water baths are a good way to rehabilitate sick reptiles and treat dysecdysis [11]. Wound healing is slow in reptiles, so stitches should be left at least 6 weeks [32]. It is best to leave stitches in place until ecdysis occurs since the increased activity in the dermis in epidermis

It should also be considered that during ecdysis, the skin becomes more perme-

Samples of the skin tissue of different reptile species were preserved in 5% formaldehyde. Pieces of different types of tissue were included in the paraffin by using usual procedure with the apparatus Leica TP1020. Histological slides were prepared before use by the procedure which ensured that histological sections adhered to the slides properly and prevented sections from falling off the slides during further procedures. Tissue samples embedded in paraffin were cut by a hand microtome (Leica) into 5-μm-thick slices, which were transferred with brushes onto the smooth surface of warm water bath (40°C) and from there on the microscopic slides. Histological slides were dried in a thermostat (50°C). For classical histological staining with hematoxylin-eosin (H&E), samples were deparaffinized in xylene substitute (Neoclear; Merck) (2 × 5 minutes) and afterward rehydrated in decreasing concentrations of ethyl alcohol (100% for 2 × 5 minutes, 96% for 5 minutes, 75% for 5 minutes) and distilled water (2 × 5 minutes). In the following steps, samples were stained with either hematoxylin (Merck) (2 minutes), washed in running water (20 minutes), stained with eosin (1–2 minutes), and washed in distilled water (5 minutes) or Toluidine blue dye solution (20 minutes) and washed in distilled water (5 minutes) three times. Further, dehydration in ethyl alcohol with increasing concentration was performed (75% for approximately 5 minutes [depending on the sample; appropriate timing is observed during staining for intensity of reaction], 96% for 1× around 5 minutes, 100% for 2 × 5 minutes). Clarification of samples after drying and staining was carried out in xylene substitute (Neoclear) (3 × 5 minutes). At the end, a drop of Neo-Mount medium (Merck) was applied onto each tissue sample and the sample was covered with cover slide. Pictures were taken on Nikon FXA microscope with Nikon

DS-F1 camera and transferred to program for image analysis by Lucia-G.

Authors would like to acknowledge financial support from ARRS, Research group P4-0053; Jasna Šporar for technical assistance with histological samples;

**152**

**Acknowledgements**

Prof. Alenka Dovč and Zlatko Golob, PhD, for their help with providing diverse reptile samples; and Catrin S. Rutland for winning the Women in Science award, which resulted in us preparing this chapter and book, and for English language proof-reading.
