Superfrost plus adhesion slides

toluidine blue O stain was used to observe the cartilage matrix. The colorant stains nuclei as blue and proteoglycans and glycosaminoglycans as light blue to violet. Cell pellets were washed twice in PBS, fixed in 10% formalin for 15 min at RT, then included in paraffin, and sliced into 5 μm sections on the SuperFrost Plus adhesion slides (Thermo Fisher Scientific) which were previously coated with poly-L-lysine (Sigma) to prevent section from detachment.
For the coloration, slides were deparaffinized and dipped in the 0.2% w/v toluidine blue O solution (the powder was provided by Serva, Germany) for 5 min at RT followed by rinsing in distilled water twice, each time for 5 min and mounted with DPX mounting medium.

Expression of Akt serine/threonine kinase 2 (Akt2) was analyzed using RISH instead of immunohistochemistry to avoid non-specific results due to the high similarities of amino acid sequences between three Akt isotypes [36 (link)]. RISH was conducted using the commercial kit (RNAscope, Advanced Cell Diagnostics, Newark, CA, USA) Concisely, FFPE tissues were sectioned into 4-μm on SuperFrost Plus adhesion slides (Thermo Fisher Scientific, Waltham, MA, USA) and were baked in 60 °C for 1 h. Tissue slides were then deparaffinized in xylene twice for 5 min each, washed in 100% ethanol twice for 1 min each, and air-dried. After deparaffinization, endogenous hydrogen peroxidase activity was quenched and slides were boiled in target retrieval solution for 15 min on a hot plate, with the temperature adjusted from 93 to 98 °C. For probe penetration, protease was applied onto the tissues and incubated in 40 °C for 30 min. Thereafter, sections were washed in distilled water, Akt2 probe (NCBI reference: NM_001012340.1) was hybridized, and the signal was amplified and developed by reacting with 3′3-diaminobenzidine. Slides were counterstained in 50% Gill’s hematoxylin. To examine whether RNA level is intact in FFPE tissues, the endogenous housekeeping gene UBC (NCBI reference: XM_038436353.1) was hybridized to serial sections of each sample.

The Forschungszentrum Jülich (Jülich, Germany) conducted all technical processing and final visualization by a company-owned software. Cryostatic sections of the brachiocephalic arteries that were 9 µm thick were generated. They were collected on SuperFrost Plus adhesion slides at −20 °C (Thermo Fisher Scientific, Dreieich, Germany). Afterwards, the samples were processed with the laser ablation system NWR 213 (New Wave Research, Fremont, CA, USA). It was linked to an inductively coupled plasma mass spectrometer (Agilent 7900, Agilent Technologies, Santa Clara, CA, USA) which performed scans with a measured spot area of 20 µm, a laser speed of 20 µm/s, and an energy level of 34%. Specimens of rat brains with a specified amount of Gd were used as standards and were measured under the same conditions to verify the accuracy of Gd quantification in harvested brachiocephalic arteries.