Citrisolv

Thionine staining was used to quantify pyramidal neuron density in the hippocampus CA1 and CA3. Slides were transported in containers filled with 0.01 M phosphate-buffered saline (PBS; pH = 7.4), then soaked in dH₂O (30 s), thionine (10 min), 50, 70, 95, and 2 × 100% ethanol (2 min each), 2 × Citrisolv (2 min each; Decon Labs, Inc., United States). Slides were covered with Permount Mounting Medium, were cover-slipped, and were left to seal overnight. For each subject, six histologically representative pictures of the CA1 and CA3 hippocampal subregions were obtained using a Leica DAS microscope with an attached SONY digital camera and were recorded via Norton Eclipse 6.0 software (Empix Imaging, Mississauga, ON, Canada). Pyramidal neuron density within 1 mm linear length was quantified with ImageJ software (National Institutes of Health, United States) by two blinded examiners with achievement of inter-rater reliability. Intact neurons were defined as having a clear nuclear area enclosing a nucleolus and a cytoplasm contained within a rounded cell body.

Tissues were collected at post-osteotomy day (POD) 0.5 to evaluate micro-damage and programmed cell death caused by surgical drilling, as well as at POD3 and POD7, when new bone formation is initiated [7 (link)]. In brief, animals were euthanized; then, the entire maxillae were dissected free from other tissues and transferred to 4% paraformaldehyde (PFA) and stored at 4 °C overnight. After fixation, samples were decalcified in ethylenediaminetetraacetic acid (EDTA), embedded in paraffin, and sectioned at an 8-μm thickness for analyses. Tissue sections were deparaffinized in Citrisolv (Decon Labs, Inc., King of Prussia, PA, USA) and hydrated via a series of decreasing concentrations of ethanol before staining or other histological/cellular activity analyses.

FFPE tissue sections (5 µm thick) were processed according to the GeoMx® Digital Spatial Profiler (DSP) protocol (NanoString Technologies, Inc.). Briefly, tissue was deparaffinized and rehydrated with CitriSolv (Decon Labs), ethanol and ddH₂O washes at room temperature. Antigen retrieval was performed in citrate buffer pH 6.0 (Millipore) at ~ 115 °C under high pressure and washed in tris-buffered saline with Tween 20 (TBS-T) (Cell signaling Technology). Tissue was blocked with Buffer W (Nanostring) for 1 h at room temperature. Slides were incubated at 4 °C overnight with antibody mixture including fluorescently tagged antibodies for IBA1-AF532 (Nanostring Technologies, Inc. #121,300,306) and CD45-AF594 (NanoString Technologies, Inc. #121,300,301) and the following oligo-tagged NanoString GeoMx human detection antibody mixtures: Immune Core Profiling, Immune Cell Typing, Immune Activation, and Cell Death (see Supplemental Material for list of detection targets). Slides were washed with TBS-T, fixed with 4% formaldehyde (Invitrogen) for 1 h at room temperature, then re-washed with TBS-T. Nuclei were stained with SYTO 13 (NanoString).

Flowers from the nine species at the three developmental stages were fixed in FAA and dehydrated in an ethanol series as previously mentioned. Samples were then cleared with CitriSolv (Decon Labs, King of Prussia, PA, USA), embedded in Paraplast Plus (Sigma-Aldrich, St. Louis, MI, USA), and stored at 4 °C. Samples were sectioned to 8 μm using a Microm HM 325 rotary microtome (Thermo Scientific, Waltham, MA, USA) and mounted on glass slides. Transverse and longitudinal sections were prepared for each species.