Animal Dissection and Histopathology

Some rats were dissected after the development of tumor masses in the parotid gland, while others were dissected after confirmation of abnormal hematology profile and enlarged viscera as a result of hepatomegaly and splenomegaly.

The rats were individually anesthetized with an oxygen-sevoflurane mixture (95–5%) for 5 min before being stabilized in a wax tray and dissected by an appointed veterinarian. All vital organs were removed, including the brain and induced masses. Organs were washed with saline and fixed in 10% buffered neutralized formalin (#NBF03-500R, Tissuepro Technology, USA) overnight at room temperature. Fixed tissue was then washed thoroughly under running tap water, rinsed with distilled water, wiped off, and instantly stored at −20°C until further processing.

The frozen tissues were brought to room temperature and then dissected by a histopathologist for tissue processing. The dissected tissues were packed into cassettes and embedded in paraffin wax using an automated tissue processor (Shandon Citadel 1000, Thermo Scientific, Cheshire, UK). Briefly, the tissues were incubated in two stations of formalin for 5 min as they were pre-fixed, followed by tissue dehydration through gradual concentrations of ethanol, starting with 70% ethanol (15 min), 80% ethanol (15 min), 90% ethanol (15 min), and finally in 100% ethanol for 15 min. Tissue sections were then cleared at two stations of xylene (20 min each) before being infiltrated with paraffin wax.

Wax-infiltrated sections were then transferred to a tissue-embedding center containing molten paraffin wax (TES 99, Medite Medical GmbH, Germany). The cassette was disassembled, the tissue section was properly oriented by the histopathologist, and topped with paraffin wax. The whole cassette was then transferred to a cold plate to solidify and form a block and then stored overnight in the fridge at 4°C.

Blocks were sectioned at 5 mm thickness using a rotary microtome (Accu-cut^® SRM™, Sakura Finetek Europe, the Netherlands). The section ribbons were then placed in a water bath and attached onto slides. The slides were then placed on a hot plate to enhance the attachment.

The slides that were fixed on the hot plate following block sectioning were first dewaxed by immersing them in two successive stations of xylene (#9990501, Shandon, Thermo Scientific, Cheshire, UK); each for 5 min. Next, the slides were hydrated in 100% ethanol for 3 min, followed by immersion in 95% and 80% ethanol; each for 3 min, and then the slides were immersed in 70% ethanol for 3 min. After rehydration, the slides were washed for two minutes in distilled water.

The hydrated slides were stained with Harris hematoxylin (#6765003, Shandon, Thermo Scientific, Cheshire, UK) for 5 min and then washed for 1 min in distilled water. The sections were then placed under running tap water for 10 min to allow the bluing development of the stain. Slides were then washed in distilled water for 1 min and placed in eosin Y (#6766008, Shandon, Thermo Scientific, Cheshire, UK) for 3 min. Next, the slides were dehydrated in 70% ethanol (1 min), then in 80% ethanol (1 min), followed by 95% ethanol (1 min), and finally in 100% ethanol for 1 min. The dehydrated slides were then cleared with two changes of xylene (2 min each), mounted with Consul-Mount (Consul-Mount, #9990440, Shandon, Thermo Scientific, Cheshire, UK), and covered with a coverslip.

The rehydrated slides were stained with the modified Masson’s trichrome stain kit for visualization of the collagenous fibers according to the manufacturer’s instructions (#MMT-M-001-50, Molequle-on, Auckland, New Zealand). Briefly, slides were deparaffinized and hydrated before being immersed in the Bouin’s reagent (which was preheated to 56°C) for 60 min. Slides were left to cool down for 10 minutes before being rinsed in tap water until sections are clear. This was followed by another rinse in distilled water. Subsequently, slides were stained for 5 min in a fresh working solution of Weigert’s Iron, Hematoxylin reagent (prepared by mixing equal parts of Weigert’s (A) and Weigert’s (B) reagents). Then, slides were rinsed in tap water for 2 min, and Biebrich Scarlet/Acid Fuchsin Solution was applied to the slides for 15 min. Next, slides were rinsed in distilled water, and the differential solution of phosphomolybdic/Phosphotungstic acid was applied to the slides for 10‐15 min or until collagen red color disappears. Aniline Blue Solution was then applied to the slides for 5–10 min without rinsing. Slides were then rinsed in distilled water, and 1% acetic acid was applied for 3–5 min. Next, slides were dehydrated in two successive changes of 95% ethanol, followed by two changes of 100% ethanol, then slides were cleared in xylene and mounted for visualization. Collagen is stained in blue, keratin and cytoplasm are red, and nuclei in blue/black.