Dmls microscope

An additional aliquot of whole blood from each snake was used to prepare blood smears stained with hematoxylin and eosin. These samples were examined for Heinz bodies and hemolysis using a Leica DMLS microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). MetHb is converted into irreversible hemichromes within erythrocytes that lead to the precipitation of hemoglobin in the form of Heinz bodies^{37 (link)}. Heinz bodies cause lysis of erythrocytes by damaging the cell membrane^{38 (link)} and cause extravascular hemolysis when erythrocytes containing Heinz bodies are phagocytosed by macrophages³⁹ . These effects result in hemolytic anemia, further adding to the hypoxia-producing effects of methemoglobin.
The remaining blood sampled from each snake.was centrifuged for 3 min (3000 rpm at room temperature) and the plasma stored at −20 °C. Plasma samples were transported frozen to the NWRC in Fort Collins, Colorado, and stored at −80 °C prior to conducting biochemical analyses.

Morphometric changes were assessed in four hemimaxillae obtained from four animals in each group. Serial parasagittal sections (4 μm) were mounted on slides and stained with hematoxylin and eosin (H&E). Three sections were selected from each tooth. Histometric analysis consisted of determining the area of unmineralized tissue in the furcation region. In the furcation region, the delimitation of the area was according to Duarte et al. [24 (link)]. The area of interest included a 1,000-μm zone under the furcation roof in the interradicular region of the maxillary first molar. The total area of the interradicular region and the area of bony mineralized tissue were measured. To determine the area of unmineralized tissue, the bone tissue area was subtracted from the total area. The area of unmineralized tissue was then expressed as a percentage of the total area. The analysis was performed by a blinded and calibrated examiner using a Leica DMLS microscope (Leica microsystems, Wetzlar, Germany; ×200 magnification). Areas of interest were selected and photographed using a Leica DFC 300 FX digital camera (Leica microsystems). The photographs taken were archived in TIFF format. Histometric analysis was performed using the Image J 1.37b image analysis system (National Institutes of Health, Bethesda, MD, USA).

FJB was used to stain brain cells under degeneration [79 (link)]. Tissue 8 µm-thick sections were deparaffinized and hydrated. Slides were incubated in 80% EtOH-1% NaOH for 5 min, followed by 2 min in 70% ethanol and 2 min in distilled water. Sections were immersed in 0.06% K₂MnO₄ for 20 min, after distilled water washes, slides were incubated at 4 °C overnight in 0.001% Fluoro-Jade B (Millipore, AG310) previously diluted in 0.1% acetic acid [80 (link)]. Slides were dried, immersed in xylene and mounted with Eco-Mount (Biocare Medical, EM897L). Images were captured at 40× magnification on a Leica DM LS microscope with epifluorescence (Leica Microsystems, Wetzlar, Germany).