Hematoxylin solution

The specimen sections were deparaffinized and rehydrated. Then, endogenous peroxidase was blocked using a hydrogen peroxide block (Abcam, UK). The specimens were washed, and antigen retrieval was performed by heating in a 10 mM citrate buffer. After protein block solution (Abcam, UK) was applied to reduce nonspecific background staining, the specimens were incubated with primary antibodies overnight at room temperature in a humidified chamber. The antigen-antibody complex was then detected using a Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam, UK), following the manufacturer's instructions. The sections were counterstained with hematoxylin solution (Mayer's modified, Abcam, UK) and dehydrated before mounting with mounting medium (Abcam, UK). As a negative control, the primary antibody was replaced with SignalStain antibody diluent (Cell Signaling Technology, USA). The stained specimens were examined under a Motic BA210 microscope.

For the H&E staining of the skin, paraffin sections were prepared by securing the skin from the epidermis to the dermis layer. The sections were then mounted on individual slides. The paraffin sections on each slide were dewaxed three times for 15 min each using xylene; hydrated in 100%, 90%, 80%, and 70% EtOH for 2 min each; and treated with a hematoxylin solution (Abcam, Cambridge, MA, USA) for about 10 min. The sections were dyed and then washed three to five times with distilled water. The sections were soaked in an eosin Y solution for 90 s and then washed with distilled water three to five times. Stained sections were immersed in 70, 80, 90, and 100% EtOH for 2 min to dehydrate the tissue; immersed three times in new xylene for 10 min each; dried at room temperature; and mounted with a mount solution. After completely solidifying the mount solution, growth and telogen HFs were observed using a phase-contrast optical microscope.

The animals were euthanized using CO₂ asphyxiation and the left whole eyes were harvested for histological analysis to demonstrate PMN infiltration and hemagiectasis in the mouse conjunctiva. The tissues were then processed for paraffin embedding and sectioning (5 µm) using microtome (Leica, Wetzlar, Germany). The paraffin sections underwent dewaxing using xylene I and xylene II, followed by dehydration using anhydrous ethanol I, anhydrous ethanol II, and 75% alcohol. Subsequently, the sections were stained with hematoxylin solution (Abcam, Cambridge, UK) for 3–5 min, rinsed with tap water, underwent liquid differentiation, returned to blue, and rinsed again. Dehydration was performed using 85% and 95% gradient alcohol, followed by staining with eosin dye solution (Abcam, Cambridge, UK) for 5 min. After staining, the sections were cleared with xylene and sealed with neutral gel for microscopic examination. The PMN cell number and hemangiectasis in the conjunctiva were measured using ImageJ software (https://imagej.nih.gov/ij/ accessed on 4 January 2024 developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).