Confocal high-resolution acquisitions were carried out on an inverted E2000U Eclipse microscope equipped with benchtop laser system and digital image C1 software (Nikon). out by ANOVA. Results Toluidine blue staining identified metachromatic intact or degranulated mast cells in the area below the palisades Vogt (Ck-3/12-positive epithelium and underneath p63 immunoreactivity). Tryptase immunoreactivity was observed close to palisades Vogt, whereas no specific signal was detected for chymase. Tryptase/AA1 transcripts were quantified in limbal and conjunctival RNA extracts, whereas no specific AM095 free base amplification was detected in corneal ones. Few mediators were overexpressed in limbal extracts with respect to corneal (Neural cell adhesion molecule (NCAM), Intercellular adhesion molecule 3 (ICAM3), Brain-derived Neurotrophic factor (BDNF), and neurotrophin 3 (NT3); < 0.00083) and conjunctival (NCAM, ICAM3, and NT3; < 0.05) protein extracts. A trend to an increase was observed for Nerve Growth Factor (NGF) in limbal extracts (> 0.05). Conclusions The specific observation of tryptase phenotype and the interesting protein signature of microenvironment (adhesion molecules, growth factors, and neurotrophins), known to partake mast cell behavior, at least in other areas, would provide additional information to better understand this crucial zone in the framework of ocular surface healthiness. = 26; 60- to 77-year-old donors; Biology and Pathology of the Eye, Prague, Czech Republic). All procedures for corneoscleral tissue handling followed the standards of the Ethics Committees of the General Teaching Hospital and the First Faculty of Medicine of Charles University, Prague, Czech Republic. Overall, experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology guidelines and adherent to the tenets of the Declaration of Helsinki concerning human subject contribution. Only corneoscleral specimens not suitable for transplantation and showing intact epithelium (from cornea to limbus and conjunctiva) as well as stored for fewer than 4 days in Optisol/Epilife medium (Bausch & Lomb, Rochester, AM095 free base NY, USA) were selected for the study. Specimens were cut in squares and quickly frozen in Optic Cutting Temperature (OCT) compound (TissueTek; Leica, Heidelberg, Germany) or snap-frozen. All specimens were sent by courier to the laboratory, according to the triple packaging/shipping procedure. Reagents Unless specified below, sterile RNAse-free plasticware and molecular/analytical-grade reagents were from Starlab (Ahrensburg, Germany), ICN (Costa Mesa, CA, USA), SERVA (Weidelberg, Germany), Sigma-Aldrich (Milan, Italy), and Euroclone (Milan, Italy) unless otherwise specified in the text. Ultrapure RNAse-free MilliQ-Grade water was provided daily (Direct Q5 apparatus; Millipore, Vimodrone, Milan, Italy) for biochemical studies and for molecular analysis as Diethyl pyrocarbonate (DEPC)-treated and autoclave aliquots. Light and Fluorescent ALPHA-RLC Microscopy OCT-embedded corneoscleral specimens were cut in 5-m serial sections (CM3050 cryostat; Leica Microsystems, Rijswijk, Netherlands), placed onto glass slides (BDH, Milan, Italy), quickly air-dried, and stored until specific staining. Sections were postfixed in cold 0.05% buffered formaldehyde and used as reported below. Sections from paraffin-embedded specimens were used for basal histology after dewaxing and rehydrating steps (downscaling Et-OH steps until water and buffered saline). Basal Histology Sections were stained/counterstained with 1% toluidine blue in 1% saline (TB), hematoxylin and eosin (HE), or cresyl violet (all from Bio-Optica, Milan, Italy), and digital images were produced with a direct E400 Eclipse light microscope (Nikon, Tokyo, Japan). Immunohistochemistry and Immunofluorescence Antigen retrieval (0.05% trypsin-EDTA solution, 2 minutes) and avidin-blocking/permeabilizing (1% BSA and 0.5% Triton X100 in PBS, 5 minutes) steps were performed before probing with specific monoclonal/polyclonal antibodies (Table?1). The Avidin-Biotin Complex technique (Vectastain Elite kit; ABC-HRP Kit; Vector Laboratories, Burlingame, CA, USA) coupled to AM095 free base 3,3-Diaminobenzidine (DAB) (Dako, Carpinteria, CA) developing was used for immunohistochemistry. Hematoxylin (Bio-Optica) counterstaining was used to better visualize the positive brown cells. Specifically, for primary antibodies developed in mouse, a mouse-on-mouse biotinylated anti-mouse Ig kit (MOM; Vector Laboratories) was used to discriminate immunoreactivity. The secondary Cy2 (green)CCy3 (red)CCy5 (blue) conjugated species-specific antibodies (1:150C1:300; donkey; Jackson ImmunoResearch, Europe Ltd, Suffolk, UK) were used for immunofluorescent labeling. Depending on the double-fluorophore mixture, nuclear counterstaining was performed with propidium iodide (PI; Molecular Probes, Eugene, OR, USA) or 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA). Confocal high-resolution acquisitions were carried out on an inverted E2000U Eclipse microscope equipped with benchtop laser system and digital image C1 software (Nikon). Channel series were performed using the negative controls (omission of primary antibodies) to reduce not specific signals. Epifluorescence acquisitions were also carried out with a direct Ni E200 Eclipse microscope.