Bx43f light microscope

Extraocular tissues were excised immediately after enucleation, to ensure proper penetration of the fixative. Then, the eyes underwent a two-step fixation process. For this purpose, a 10% formaldehyde solution was utilized, at ten times the volume of the tissue being studied. For the first 24 h, the ocular globes were submerged in the fixation solution, as a whole. Subsequently, they were briefly removed from the fixative, and sectioned in half, along the anatomical sagittal plane (with the blade placed perpendicular to the superior and inferior rectus muscles). Then, the obtained halves were fixed for a second 24 h period, in the same solution. Through employing a preliminary fixation of the entire globe, collapse upon halving was avoided. Ultimately, samples were embedded in paraffin and sectioned at 5 μ, then stained with haematoxylin-eosin and examined using an Olympus BX43F light microscope (Olympus, Tokyo, Japan). Pictures of representative areas were captured using the microscope mounted camera (Olympus UC30 camera with the Olympus U-CMAD3 adapter).

Following cell suspension attachment, microscope slides were heated to 95 °C for 25 min. Then slides were immersed for 20 s in Sörensen phosphate buffer (SPB) containing trypsin (2 µg/mL; pH 6.8). The action of trypsin was stopped with subsequent immersion of slides for 30 s into SPB containing fetal bovine serum (10%; pH 6.8), rinsed in SPB and allowed to air dry. Wright staining solution (WSS: Hydrion buffer capsules diluted in dH₂0, pH 6.8 (cat. #60784–226, Micro Essential Laboratory, New York, NY, USA) and Wright stain, 3:1 ratio) was prepared. The slides were placed in a horizonal position on a staining rack and entirely covered with WSS for 5 min. After washing with distilled water, slides were drained and allowed to air dry. At least 10 metaphase spreads of chromosomes per passage were viewed under an Olympus BX43F light microscope at 1000× magnification.

Both p16^INK4A and HSV immunohistochemical diagnosis was performed by an experienced certified pathologist, head of the Department of Pathomorphological and Oncological Cytology (A.H.), but the intensity of the immunohistochemical reaction was estimated independently by two pathologists as described in our previous work [33 (link)]. Tissue specimens were histologically verified to confirm the diagnosis, and histological type and the diagnosis was performed after the verification of Hematoxylin – Eosin staining. An Olympus BX43F light microscope (Olympus America, Inc., Melville, NY, USA) was used for the evaluation of slides.
Proteins expression in tissues was assessed semi quantitatively, considering the intensity of immunostaining and the number of cells showing immunoreactivity for the analyzed proteins. The expression of p16^INK4A for HPV and HSV proteins was evaluated in FFPE samples according to a modified Remmele scale [34 (link), 35 (link)] by combining the intensity of staining and pattern of distribution. Expression of p16^INK4A proteins was graded based on reaction intensity juxtaposed with the staining intensity of positive controls. The evaluation included 2 parameters of protein expression:

Two types of pattern distribution in expressed results:

Focal,

Diffused.

Intensity of staining:

Int 1 = weak,

Int 2 = moderate,

Int 3 = strong.

On the final day of drug administration, the tissues were collected and examined by H&E staining, Oil Red O staining and Picrosirius Red staining as previously described 33 , 34 (link). In brief, hepatic tissues were fixed in 4% PFA, embedded in paraffin and cut into the sections. Paraffin-embedded sections were dewaxed, rehydrated and stained with H&E stain or Picrosirius Red stain. For Oil Red O staining, the livers were fixed in 4% PFA, dehydrated in 30% sucrose. After frozen at -20 °C, the liver tissues were cut into the sections at 12 μm thickness. The liver sections were incubated in 0.2% Oil Red O reagent for 20 min. The images were acquired on Olympus BX43F light microscope (Tokyo, Japan).