Toluidine blue

Three replicate ovary and three replicate hypanthium tissue samples were dissected from mature (132 DAT) GA₃-treated and HP fruit and embedded for sectioning^{41 (link)}. Briefly, tissue was fixed in 4% paraformaldehyde (Affymetrix USB, OH) for 1e week, dehydrated through an ethanol series, and embedded in Paraplast Plus paraffin wax (Sigma-Aldrich, MO). Embedded tissue was sectioned at 8 µm with an 820 rotary microtome (American Optical, NY) and mounted on Superfrost plus microscope slides (Thermo Fisher Scientific, MA). Sections were washed with Histo-Clear II (National Diagnostics, GA), rehydrated and stained with 0.05% Toluidine Blue (Electron Microscopy Sciences, PA). Stained slides were viewed at 10x on an Optiphot-2 light microscope (Nikon, JPN); digital images containing ~1.3 × 1 mm spans of the largest observed cells were taken with a PAXcam (MIS Inc, IL). Three images from each replicate were analyzed by counting and measuring cells using ImageJ^{42 (link)}. Cell size was averaged for nine images per tissue type for each treatment and compared statistically between treatments using an unpaired-t test.

Eyes were enucleated, put in a plate with cold PBS and an incision was made under the microscope to let the fixative enter into the eye. Eyes were immersed in the fixative solution 2 h at RT while rotating and after taking out the lens, eyes were put on fresh fixative o/n at 4 °C in (2% PFA, 2% GA (Sigma, #G5882) in 0.1 M cacodilate buffer ph7.4 (CB, Sigma, #C4945). Next day we washed abundantly in 0.1 M CB (3 times for 15 minutes). Then, eyes were put under rotation in the solution 1% Osmium tetroxide, OsO_4, (Sigma, #75632, 1% Potassium ferrycianide (Sigma, #702587) diluted in distilled water for 2 h at 4 °C in dark. Next, they were washed abundantly with water (until no yellow color in detectable). Then, we dehydrate the samples following 50%, 70%, 80%, 90%, 95%, and twice 100% for 10 min each in shaker. Finally, we underwent the inclusion process by incubating 3 times in propylene oxyde (Sigma, #540048) for 10 min. To infiltrate the tissue with the resin Epon (EMS:Embed 812 #14120), we use propylene oxide:Epon resin 3:1 (1 h), propylene oxide:Epon resin 1:1 (overnight), propylene oxide:Epon resin 1:3 (1 h) and pure Epon resin 2 times for 1 h, all in the shaker at RT. Finally, we included the eyes oriented in Epon in the mold in the oven at 60 °C for 12 h. For visualization, 0.5 µm-semithin sections were stained with toluidine blue (Electron Microscopy Sciences, #22050).

At the conclusion of HBP each day horses were nebulized with 0.005 mg/kg albuterol (albuterol sulfate 0.083%, Nephron Pharmaceuticals Corporation, West Columbia, SC) and allowed a 15-min waiting period to reduce any residual bronchoconstrictive effects of histamine prior to BAL. Horses were then sedated (xylazine 0.5 mg/kg IV, butorphanol 0.01 mg/kg IV) for BAL, as previously described, to assess the effect of nebulized lidocaine on the cellular inflammatory response. BALF was kept on ice until it was delivered to the laboratory within 30 min, where it was centrifuged at 500 g for 10 min at 4°C. Slides were then prepared using the sedimentation method and stained with modified Wright-Giemsa (Dip Quik, Jorgensen Laboratories, Loveland, CO) and toluidine blue (Electron Microscopy Sciences, Hatfield, PA) stains, the latter for the enumeration of mast cells. Cells (n = 500 Wright-Giemsa, n = 1,000 toluidine blue) were classified by two investigators (JM, MM) as the percentage of total cells that were macrophages, lymphocytes, neutrophils, eosinophils, and mast cells (400x magnification). Horses were determined to have an abnormal BALF cytology using cutoff values established by our laboratory (> 10% neutrophils, > 2% mast cells, or ≥ 1% eosinophils) (24 (link)).