Shandon excelsior es tissue processor

Mouse ScWAT biopsies were fixed overnight in 10% neutral buffered formalin at 4 °C and wax embedded in Surgipath Formula “R” paraffin using the Shandon Excelsior ES Tissue processor (Thermo Fisher Scientific). Tissues were sliced into 3 µM sections on a microtome and adhered to super adhesive slides by overnight incubation at 60 °C. Slides were rehydrated and underwent deparaffinization by antigen retrieval (sodium citrate buffer; 10 mM sodium citrate, 0.05% Tween20, pH 6). Sections were treated with 3% H₂O₂ for 10 min and blocked with 2% BSA in TBST for 30 min. Sections were subsequently incubated in primary antibody (Table 1) at 4 °C overnight, followed by incubation with Alexa Fluor 488 anti-rabbit or 555 anti-mouse secondary antibody for 1 h at RT (1:1000; Fisher Scientific). Slides were mounted using super gold anti-fading mounting media with DAPI (Vector Laboratories, Burlingame, CA, USA) to visualize the nuclei. Images were visualised and captured on EVOS AUTO microscope (Thermo Fisher Scientific).
3T3-L1 cells were grown on 8 chamber slides, fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 10 min, permeabilised in 0.2% Triton-X 100 (Sigma, Welwyn Garden City, UK) and blocked with 2% BSA in TBST for 30 min.

Endometrial biopsies were fixed overnight in 10% neutral buffered formalin at 4°C and wax embedded in Surgipath Formula ‘R’ paraffin using the Shandon Excelsior ES Tissue processor (ThermoFisher). Tissues were sliced into 3 μM sections on a microtome and adhered to coverslips by overnight incubation at 60°C. Deparaffinization, antigen retrieval (pH 9), antibody staining, haematoxylin counter stain and 3,3’-diaminobenzidine colour development were fully automated in a Leica BondMax autostainer (Leica BioSystems). Tissue sections were stained for CD56 (a uNK cell-specific surface antigen) using a 1:200 dilution of concentrated CD56 antibody (NCL-L-CD56-504, Novocastra, Leica BioSystems). Stained slides were de-hydrated, cleared and cover-slipped in a Tissue-Tek Prisma Automated Slide Stainer, model 6134 (Sakura Flinetek Inc., CA, USA). Bright-field images were obtained on a Mirax Midi slide scanner using a 20× objective lens and opened in Panoramic Viewer v1.15.4 (3DHISTECH Ltd, Budapest, Hungary).

Endometrial biopsies were fixed overnight in 10% neutral buffered formalin at 4°C and wax embedded in Surgipath Formula ‘R’ paraffin using the Shandon Excelsior ES Tissue processor (Thermo Fisher). Tissues were sliced into 3 μm sections on a microtome and adhered to coverslips by overnight incubation at 60°C. Deparaffinization and rehydration were performed through xylene, 100% isopropanol, 70% isopropanol, and distilled water incubations. Following antigen retrieval, slides were washed, blocked, and incubated in primary HTR2B antibody (1:200; Fisher Scientific) overnight at 4°C. After washing three times, slides were incubated with Alexa Fluor 594 (1:1000; Fisher Scientific) for 2 hr, washed and mounted in ProLong Gold → Antifade Reagent with DAPI (Cell Signaling Technology). Slides were visualized using the EVOS Auto system, with imaging parameters maintained throughout image acquisition.

Dogs were anesthetized by intramuscular injection of Alfaxan (Jurox, Hunter Valley, NSW, Australia) and Rompun (Bayer, Leverkusen, Germany), and then euthanized. Necropsy was performed on all animals, with macroscopic evaluation of the thoracic and abdominal cavities and tissues. After macroscopic examination, the excised pancreas was fixed in 10% neutral buffered formalin. The three lobes of the pancreas (head, body, and tail) were sectioned and dehydrated in graded ethanol, cleared in xylene using a Shandon Excelsior ES tissue processor (Thermo Fisher Scientific), and embedded in paraffin blocks using an EG1150H paraffin-embedding station (Leica Biosystems, Wetzlar, Germany). Histological examination of the pancreas was performed by an experienced veterinary pathologist (Woo-Chan Son, Asan Medical Center, Korea), and the lesions were semiquantitatively graded as minimal (+1), slight (+2), moderate (+3), marked (+4), or severe (+5).

Formalin-fixed intestinal sections from the duodenum and jejunum were dehydrated through a series of graded ethanol baths followed by xylene in a Shandon™ Excelsior™ ES Tissue Processor (Thermo Fisher Scientific Inc., Waltham, Massachusetts), before being embedded in paraffin wax, sectioned at 4 μm and stained with hematoxylin/eosin. Histological sections were examined under a Zeiss Primostar light microscope and images were captured using ZEN imagine software (Zeiss Germany, Oberkochen, Germany). Images were viewed to measure morphometric features of the intestinal structure at 10× magnification. From sections, the villus height and the crypt depths were determined using ImageJ (NIH) software (Schneider et al., 2012 (link)). The villus height was estimated by measuring the vertical distance from the villus tip to the villus-crypt junction for 10 villi/section, and the crypt depth by the vertical distance from the villus-crypt junction to the lower limit of the crypt, for 10 corresponding crypts/section.

Liver, small bowel, splenic, and renal biopsies were divided and flash-frozen in liquid nitrogen and stored at −80°C or processed for histology, by fixing under vacuum in 10% neutral-buffered formalin for 24 h at 37°C and stored for up to 1 month. Histology samples were paraffin-embedded on a Shandon™ Excelsior™ ES tissue processor (Thermo Fisher Scientific Inc., Waltham, U.S.A.). Three millimeter sections were stained with Hematoxylin and Eosin (H & E).

Eyes were obtained immediately after the animals were euthanized as described above. The eyes were fixed for 48 hours in 4% paraformaldehyde solution, rinsed in PBS before overnight storage in 3% sucrose in PBS. Eyes were slowly dehydrated in increasing ethanol gradient and xylol baths over a period of 3 weeks before paraffinization using a Shandon Excelsior ES tissue processor (Thermo Scientific, Ghent, Belgium). Paraffin embedded tissue blocks were sliced into 7μm sections. Selected sections were deparaffinized in a decreasing series of xylol and ethanol baths before staining the section with Hematoxilin and Eosin. Image acquisition was performed on a Zeiss Imager Z1 microscope, both under normal white light and using epifluorescence in the green channel. The latter was used to assess the presence of fibrin within the thrombus as described by Lucas TC et al [29 (link)].