Histostar embedding center

By the end of the study period (Days 42), major organs (liver, spleen, and kidneys) were harvested and weighed. Both liver and spleen tissues were then immediately preserved in 10% neutral buffered formalin (NBF) for no more than 24 h. Well-preserved tissues were processed by the Epredia Excelsior AS Tissue Processor (ThermoScientific, Waltham, MA, USA) and embedded with paraffin wax by the Epredia HistoStar Embedding Center (ThermoScientific, USA). Then, the formalin-fixed paraffin-embedded (FFPE) blocks were cut into 0.5 µm thick sections by a Rotary Microtome (Leica BioSystems, GmbH, Nußloch, Germany) and mounted on glass slides before being subjected to hematoxylin and eosin (H&E) staining. The stained spleen and liver were examined by the Zeiss Axioscan 7 Automatic Slide Scanner with 40× magnification and 20× magnification. The viscera index calculation was described in [18 (link)]. The viscera index of the hamster’s major organs (liver, spleen, and kidneys) was calculated with the following equation:
(Weight of Organ/Body weight before sacrifice) × 100

Gm were infected with H37Rv (2 × 10⁶ CFU) and processed for TEM and histological analysis as described [11 (link),12 (link)]. In brief, for TEM, hemocytes of 10 larvae were collected at each time-point and washed as described for THC. Pelleted hemocytes were resuspended in 3% glutaraldehyde, fixed for 10 min, pelleted (800 g for 10 min), and stored at 4°C. Pelleted hemocytes were treated with 1% osmium tetroxide, dehydrated in ethanol and embedded in resin. Sliced sections (70–90 nm) were mounted, stained with uranyl acetate (0.5%) and lead citrate (3%), and examined using an Tecnai bioTWIN transmission electron microscope (FEI Company, USA). Healthy hemocytes from naïve larvae and suspensions of H37Rv were used as controls. For histological analysis, three larvae were fixed at each time-point by injecting 100 μl of 10% buffered formalin. Fixed larvae were cut into halves along the dorsal line. Larvae were processed for histology using the Sakura Tissue-Tek VIP (Sakura, USA), embedded in paraffin wax using the Histostar™ Embedding Center (Fischer Scientific, USA), and sliced into 4 μm sections using an RM2135 microtome (Leica Biosystems, Germany). Sections were mounted onto glass slides and processed for H&E or ZN staining and examined using an Eclipse 80i light microscope (Nikon, Japan). Uninfected larvae were fixed and used as controls.