The Use of Modified Masson’s Trichrome Stain to Recognize the Stratum Corneum in the Epidermis of the Skin and Measure It

ABSTRACT


INTRODUCTION
The layers of the skin consist of two areas: the upper part is the epidermis, and the lower part is the dermis, and under the skin there is a subcutaneous layer, the epidermis in vertebrates has three layers from the inner to the upper layer: stratum germanitivium which have clearly nuclear and are very active in mitotic, and then stratum spinosum which seems like spine shape, and last the died and scaly upper layer stratum corneum (Yousef et al., 2022).Lizards have three layers and the stratum corneum seems large and clear because of the environment that lives in it (Ahmed et al., 2016).Masson's trichrome is a classical method of visualization with several variants.The nuclear dye can be Masson's hemalum or several hematoxylin solutions.The cytoplasmic dye is fuchsine.An aniline-derived dye allows the differentiation of collagen fibers.This staining method yields good results for morphological studies because the different parts of the tissue are very well contrasted (Exbrayat, 2016).

MATERIALS AND METHODS Masson's Trichrome Stain
In the protocol staining with Masson's trichrome stain according to Luna (1968), it used Weigert's iron hematoxylin stain the nuclei in black, the acid fuchsin stains cytoplasm and muscle fibers in red then treated with phosphmolybdic acid, the aniline blue stain the collagen in blue.In routine embedding of skin, after dissection the selective region was fixative in formalin 10% to about 18-24 hrs., then washed in tape and distilled water each for 30 minutes, to dehydrate the sample used ascending of ethanol alcohol (70,80,90,95%) for 30 minutes, the sample then immersed in xylol to purify it for 30 minutes.Paraffin wax was used for the infiltration, molting it at 58-60°C for 30 minutes, embedding it in the oven, and then blocking it in a stainless steel template for sectioning with a rotary microtome at a thickness of 5 microns.

2-Weigert's Iron Hematoxylin Solution
The use of trichrome by immersion of the fixative sample in Weigert's iron hematoxylin then in three different solutions (A,B,C):

Weigert's Iron Hematoxylin Working Solution
Mix equal parts of stock solution A and B. This working solution is stable for 3 months (not good after 4 months)

5-Aniline Blue Solution
The method modified by Kiernan (2008) where samples are placed after putting them on the slides in a Coplin jar and immersed in three stages of xylol for about 4 minutes, then putting them in descending concentrations of ethanol 100%,96%, 90%, 80%, 70% for 4 minutes per once.
b. Immerse slides in Bouin's solution at 60°C for 45 minutes and wash them in tap water until the yellowish color of Bouin's solution disappears but this step is not necessary if the samples are not fixed in formalin.
c. To recognize the nuclei, immerse the slides in Weigert's hematoxylin for 8 minutes and wash them in tap water for 2 minutes.
d. Acid fuchsin is used to stain the cytoplasm and erythrocytes for 5 minutes then washed in tap water for 2 minutes.
e. Put the slides in Phosphomolybdic acid for about 10 minutes to moderate the stain.
f.To stain the collagen and fibroblast immerse the slides in methyl blue for 5 minutes.
g. Wash the slides in tap water for 2 minutes.h.At last, put the slides in 1% glacial acetic acid for 1 minute.
j. Put the slides in xylol for 1 minute.k.Mount the coverslips by DPX on the slides and examine them with a light microscope.

Modified Masson's Trichrome 1-Preparation
The normal formation of the prepared tissues to be stained with Masson's trichrome is formalin as a fixative solution in 10% concentration, but in this modified method the fixative solution is Carnoy's which is used in fixative of neural tissues and glycogen.The Carnoy's solution is prepared as follows: Glacial acetic acid 10 ml Chloroform 30 ml Absolute ethanol alcohol 60 ml The Fixation period is about 30 minutes to 1 hour, tt will shrink if it is placed more than this time.The preparation steps will be carried out after fixation normally and the sample is sectioned with a thickness of 5 microns.

2-The Staining
1-The methods of staining are done normally.First, the slides are put in Coplin jar and immersed in three stages of xylol for about 4 minutes and then they are placed in descending concentrations of ethanol 100%,96%, 90%, 80%, 70% for 4 minutes per once.
2-The slides were immersed in Bouin's solution at 60°C for 45 minutes and washed in tap water until the yellowish color of Bouin's solution disappeared (but this step is not necessary if the samples are not fixed in formalin).In this modified method we used Bouin's solution although formalin was not used.
3-The steps of staining were then done as normal Masson's trichrome stain.

RESULTS AND DISCUSSION
The epidermis of lizards stained with Masson's trichrome showed three colors, the stratum germanitivium appeared with blue to gray nuclei and red cytoplasm, the middle region of the epidermis the stratum spinosum is seen red in the upper region the stratum corneum, in normal Masson's trichrome seen red, the upper two layers are not recognizable, and the Bouin's solution colored the corneum in yellowish color, This result makes the Bouin's solution color the stratum corneum with yellow color and can be identified and measured easily compared to the hematoxylin and eosin stain method and the normal Masson's trichrome method (fig 1 -A,B,C).et al. (2011) in the contain of Bouin's fluid of picric acid that gives the fixative samples suitable for staining by trichrome stains, the picric acid slow penetrating that precipitates proteins and forming salts (picrate) with proteins and it associated with yellow staining to bright.This study uses of carnoy's fixative because the formalin if fixed in the specimens for a long time causes the formation of formic acid and precipitate formalin pigments in the histological section and it agrees with Thavarajah et al. (2012).

CONCLUSIONS
The use of bouin's fluid in this study assisted in determining the stratum corneum from other regions by staining it yellow despite washing it in tap water and alcohol and it is the novel of this research.
Lihui et al. (2011)6)ees withSinghal et al. (2016), that explains fixation with carnoy's fluid was a fast fixation with good results and is used in urgent diseases and it takes a maximum time of 6 hours, while 10% buffered formalin and bouin's solution take 18 hours in minimum.The finding agrees withLihui et al. (2011)and Bultitude

Table 1 :
The measure of stratum corneum in μm