Mcilwain tissue chopper

Two weeks after the SCT, rats were anesthetized with isoflurane and then received a lethal dose of Morbital (pentobarbital, 120 mg/kg body weight, intraperitoneal; Biowet Puławy Ltd., Puławy, Poland). Then they were transcardially perfused with ice-cold saline (IF, LMD, qPCR groups) or 4% paraformaldehyde (TEM groups). Their vertebral columns were then excised, placed on ice, and the spinal cords were dissected. Spinal cords and selected hindlimb muscles (TA, Sol) intended for qPCR analysis were cut with a McIlwain tissue chopper (Ted Pella Inc., Redding, CA, USA) to 0.8mm transverse sections, frozen on dry ice, and stored at −80 °C. In the LMD group, spinal cord L3-6 segments (where TA and Sol MNs are located) were surrounded by Jung tissue freezing medium (Leica, Nussloch, Germany) and frozen at −80 °C until they were cut into longitudinal sections, as described [9 (link)]. The muscles intended for IF were fixed in 4% paraformaldehyde in 0.1 M PBS for 60 min at room temperature (RT), then moved to a cold PBS solution and stored at 4 °C.

Visual cortex Organotypic cultures (OTCs) were prepared from P0/P1 neonate rat pups (Long Evans) as described (Hamad et al., 2014 (link)). Briefly, cortex blocks were cut into 350 μm slices with a McIlwain tissue chopper (Ted Pella, Redding, CA, USA). Slices were mounted on a coverslip with a plasma/thrombin coagulate and cultured at 37°C in roller-tubes with 700 μl semi-artificial medium containing: 25% adult horse serum, 25% Hank’s balanced Salt Solution, 50% Eagle’s Basal Medium, 1% NeuroCult™ SM1 Neuronal Supplement (STEMCELL Technologies, Cologne, Germany, Cat.# 05711), 1 mM L-Glutamine (all from Life Technologies, Karlsruhe, Germany), and 0.65% D-Glucose (Merck, Darmstadt, Germany). Excessive glial growth was prevented by treating OTCs at DIV 2 with a mix of uridine, cytosine-ß-D-arabinofuranoside, and 5-fluorodeoxyuridine (all from Sigma–Aldrich, Steinheim, Germany) for 24 h. The medium was changed every third day. OTCs from every individual animal (4–5 animals per batch) were allocated to all experimental conditions run with this batch of cultures.

Tumour tissues were sectioned into ~300 μm slices using McIlwain tissue chopper (TedPella). These tumour sections were randomized and cultured in 48-well flat bottom plates coated with stage and grade-matched TMP with RPMI medium supplemented with 2% AS, 8% FBS (Life Technologies. 10270-106), 1 × Insulin-Transferrin-Selenium (ITS, Life Technologies. 41400-045), 1 × GlutaMAX (Life Technologies. 35050-061) and 1 × penicillin, streptomycin and amphotericin B (Life Technologies. 15140-122). Tumour slices (n=3) were treated with either anti-EGF neutralizing antibody (rabbit monoclonal, clone D8A1, Cell Signaling Technology. 12157) or with TPF (for HNSCC) or with cetuximab+FOLFIRI (for CRC) or with dimethylsulphoxide (DMSO; vehicle control) for 72 h. The final concentration of DMSO was kept ≥0.01%. Media with drugs were changed every 24-h interval. A portion of each tumour slice was used for cell viability (assessed by WST) and remaining tumours were fixed in 10% buffered formalin and embedded in paraffin. The paraffin-embedded tumours were used for histological (hematoxylin and eosin stain) and IHC analysis including proliferation and cell death.

Mouse tissue were harvested and immediately homogenized with the McIlwain Tissue Chopper (Ted Pella, Inc.) to obtain a single-cell suspension as described previously [41 (link)]. Briefly, mechanically homogenized tissue was digested in RPMI 1640 medium containing 0.525 mg/mL collagenase Type 1 (Worthington Biochemical Corporation), 0.02 mg/mL Deoxyribonuclease I (Sigma), and 10% FBS in a 37°C shaker for 1 hour at 250 rpm. The homogenates were passed through a 70 μm filter and red blood cells were lysed with RBC Lysis Buffer (0.034 M aluminum chloride, 0.01 M potassium bicarbonate, 0.1 mM EDTA in dH₂O) for 30 sec. Cells were suspended in flow wash buffer (1% BSA and 0.1% sodium azide in DPBS) and an equal number of cells subjected to flow cytometry. Cells were first incubated with Chrom Pure Mouse IgG (Jackson Immuno Research) to block Fcγ receptors. Cells were then stained with extracellular antigen antibody on ice for 30 minutes prior to permeabilization/fixation on ice for 20 minutes and intracellular staining on ice for an additional 30 minutes. Cells were washed and measured with the BD LSR II flow cytometer (BD Biosciences). Data was analyzed using FCS3 Express software (De Novo).

Rats were humanely euthanized, brains were rapidly removed (within 30 s of decapitation) and immersed in ice-cold (4–5 °C) artificial cerebrospinal fluid (aCSF; 120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl₂, 1 mM MgCl₂, 0.4 mM KH₂PO₄, 5 mM HEPES, and 10 mM D-glucose; pH 7.4) that had been oxygenated for 1 h using 95% O₂:5% CO₂. Coronal sections (200 μm) were prepared using a modified McIlwain tissue chopper (Ted Pella. Inc.; Redding, CA) with a chilled stage and blade, then transferred to a holding chamber containing continuously oxygenated aCSF at room temperature (~ 23 °C).

RT‐PCR, experiments were performed on two groups of female C57BL6 mice: young (P30, n = 4) and middle‐aged (13 month‐old, n = 4). Animals were anesthetized with isoflurane and decapitated and hippocampi were isolated and sliced using a McIlwain tissue chopper (TedPella, Redding, California, USA; slices were 300 µm thick), as described previously (Ting, Daigle, Chen, & Feng, 2014). The slices were placed under stereoscope (Optatech, Warszawa, Poland) and CA1 hippocampal layers were dissected with a tip of a needle (0.3 × 8 mm) and the region of stratum pyramidale of each slice was absorbed directly to TRI Reagent‐containing (Applied Biosystems, Waltham, Massachusetts, USA) syringe. From each animal, 5–10 dissected stratum pyramidale samples were collected (Supporting Information, Figure S1).