Leica cm1950 cryostat

C57BL/6J or Tlr4^−/− mice were injected with vincristine (i.pl., 10 μg, 100 ng or i.p., 0.5 mg/kg) or with vehicle (i.pl., 5% glucose or i.p., PBS) using the injection schedule shown in Figure 1. After 24 h or on day seven, the mice were anesthetized with 10 mg/kg xylazine and 100 mg/kg ketamine i.p. and transcardially perfused with ice-cold PBS followed by ice-cold 4% paraformaldehyde in PBS (Sigma-Aldrich). The injected hind paws were dissected and post fixed for 16 h in 4% paraformaldehyde at 4°C. The paws were incubated at 37°C in decalcification solution containing 20% EDTA and 3% citric acid (both Sigma-Aldrich), pH 7.2 for 14 days followed by 48 h incubation in 50% Organic Compound Tissue (OCT, Tissue – Tek) in PBS. Finally, the paws were frozen in OCT (Tissue-Tek). Tissue was cut with a Leica cryostat CM1950 (Leica Biosystems). The paws were cut into 5 μm thick sections and were stained with hematoxylin and eosin in a Leica ST5020 Autostainer (Leica Biosystems, Mt Waverly, VIC, Australia). Pictures were obtained using the wide-field Zeiss AxioImage M2.m Microscope and Axiocam 506 camera. Three animals per group and 5 sections per animal were analyzed according to the scoring criteria detailed in Supplementary Table S5 by a blinded veterinary pathologist. A representative picture for each group is shown.

Hind legs of mice at the age of 14 weeks (young mice with or without forced exercise on a treadmill) as well as 9 months, 12 months, and 18 months (ageing mice without additional application of stress) were dissected. For paraffin-embedding, legs were fixed with 4% PFA in PBS at 4 °C overnight and decalcified in 20% ethylene diamine tetraacetic acid (EDTA) with 6.6% (w/v) Tris under rotation for 2 weeks at RT. During this time, the decalcifying solution was changed every 2 days. Subsequently, legs were dehydrated by a graded series of ethanol and isopropanol solutions and embedded in paraffin (Paraplast, X880.1, Roth, Germany). Moreover, 4.5-µm-thick sections were cut through the frontal plane of the knee joint with a rotation microtome AM355 (Microm, Wetzlar, Germany) and collected on glass slides. For the preparation of cryosections, dissected unfixed hind legs were embedded in O.C.T. compound mounting medium (00411243, VWR chemicals, Darmstadt, Germany), frozen in liquid nitrogen, and cut through the sagittal plane of the knee joint with a Leica CM1950 cryostat (Leica Biosystems, Wetzlar, Germany). Lastly, 4.5-µm-thick cryosections were collected on crystal clear book repair adhesive tape [57 (link)].

Knees from 14-week-old WT and ERp57 cKO mice were dissected, embedded in O.C.T. compound mounting medium (00411243, VWR chemicals, Darmstadt, Germany), and frozen in liquid nitrogen. A Leica CM1950 cryostat (Leica Biosystems, Wetzlar, Germany) was used to generate 20 µm-thick frozen tissue sections. Sections were thawed, submerged in PBS buffer, and analyzed with a NanoWizard I AFM (JPK Instruments, Berlin, Germany) in combination with an inverse optical microscope (Axiovert 200, Zeiss, Göttingen, Germany) as described before [23 (link),54 (link),55 (link)]. Briefly, silicon nitride cantilevers (0.1 N/m, MLCT, Bruker) were used to record 625 indentation curves in an area of 3 × 3 µm². For each sample, 9 areas were measured. The sample stiffness was determined by fitting a modified Hertz model to the force-indentation curves using the JPK Data Processing software (V5.0.96, JPK Instruments, Berlin, Germany). Histograms of the Youngs Moduli were generated and a linear combination of two Gaussian functions was fitted to the histograms utilizing Igor Pro software (Version 6.3.7.2, WaveMetrics).

Ly6C^high monocytes from db/db or db/+ mice were label with PKH26, resuspended in PBS at 4 × 10⁵ viable cells/0.2 ml, and injected into the jugular vein of groups of recipient db/db mice. One week later, one set of mice were analyzed for tissue infiltration. The liver and kidneys were collected, cryopreserved in OCT compound, cut into 10-μm-thick sections on a Leica CM1950 cryostat (Leica Biosystems, Nussloch, Germany), and mounted on slides. Adipose tissue was collected, cryopreserved in super cryoembedding medium (SCEM-(L1)), cut into 50-μm-thick sections, and mounted. PKH26+ cells were detected by fluorescence microscopy (model BZ-X700; Keyence, Osaka, Japan) at excitation and emission wavelengths of 551 and 567 nm, respectively. For quantification, the number of cells in 16 high-power fields per sample were counted.
Body weight, IPGTT, IPITT, and levels of blood glucose, serum fructosamine, urinary glucose, urinary albumin, and urinary 8-hdroxy-2′-deoxyguanosine (8-OHdG), were monitored in additional groups of db/db recipient mice at various times over 4 weeks after cell transfer, as described below.