Adhesive silane coated glass slide

MI model rats were sacrificed under ether anesthesia. Representative cardiac tissue specimens were harvested immediately, embedded in optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA, USA), and frozen in hexane (Wako Pure Chemical Industries, Osaka, Japan). The sections (5 μm) were prepared using a cryotome, MICROM HM560 (Carl Zeiss, Jena, Germany), at a temperature of − 20 °C and mounted on adhesive silane-coated glass slides (Matsunami Glass Industries, Kyoto, Japan). In accordance with the established procedure [21 (link)], the sections were then pre-incubated in 50 mM Tris buffer (pH 7.4) at room temperature for 20 min followed by incubation in the same buffer containing [¹⁸F]FEDAC (18 MBq/L) at room temperature for 30 min. After incubation, the sections were washed twice for 2 min each in 50 mM cold fresh Tris–HCl buffer and for 10 s in distilled water. They were then dried with a warm air current, placed in contact with an imaging plate (BAS-MS 2325; Fujifilm, Tokyo, Japan) for 60 min, and analyzed using a Bio Imaging Analyzer System (BAS 5000; Fujifilm).

Animals were transcardially perfused with 4% PFA in PBS. Spinal cord and brain tissues were post-fixed with 4% PFA in PBS at 4 °C overnight, cryoprotected in 30% sucrose in PBS, and then embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA) for frozen sectioning. Coronal sections were cut at 30-μm thickness on a cryostat and mounted on Matsunami adhesive silane-coated glass slides (Matsunami Glass, Osaka, Japan).
For histology, sections were stained with cresyl violet (Nissl stain; Sigma). Brain lesion volume was estimated by measurement of the area of lost tissue in each of three to five sections spaced 0.5 mm apart. The total lesion volume was calculated as described previously.^{36 (link)}For immunohistochemistry, sections were permeabilized in PBS containing 0.1% X-100 and 0.5% BSA for 1 h at room temperature. Sections were then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at room temperature. The primary antibodies used were as follows: mouse anti-NeuN (1 : 100; Merck, Cat# MAB377, RRID: AB_11210778), rabbit anti-PHD2 (1 : 200; Abcam), rabbit anti-PKCγ (1 : 100; Santa Cruz, Cat # sc-211, RRID: AB_632234). Alexa Fluor 488- and 588-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibodies (1 : 500; Invitrogen) were used as secondary antibodies.

The tissue blocks were sliced to a thickness of 10 μm at -20°C using a cryostat (CM1950; Leica, Wetzler, Germany). Serial tissue sections were mounted on an indium tin oxide-coated glass slide (Bruker Daltonics, Bremen, Germany) and a Matsunami adhesive silane-coated glass slide (Matsunami, Osaka, Japan) for IMS analysis and hematoxylin and eosin (HE) staining, respectively. Before analysis by IMS, the pathologist confirmed that the tumor and non-tumor regions of parotid cancer were present in a single section using HE-stained images of serial tissue sections. The non-tumor areas confirmed to be normal parotid tissue were included in the analysis. Regions with insufficient pathological findings were excluded from the analysis.
For IMS, each tissue section was coated with 9-aminoacridine (Merck, Darmstadt, Germany), which served as the matrix. Each slide was coated with a 9-aminoacridine matrix layer obtained by sublimation at 220°C, and the thickness was set to 1 μm using IMLayer (Shimadzu Corporation, Kyoto, Japan).

To assess the purity of PT cells, immunocytochemistry for megalin, a brush‐border protein in PT cells, was used. Isolated PT cells and DT cells were centrifuged, and each pellet was smeared on a Matsunami adhesive silane‐coated glass slide (Matsunami Glass Ind., Ltd., Osaka, Japan). PT and DT cells were fixed with 2% paraformaldehyde for 10 min and then incubated with 10% donkey serum at room temperature for 60 min. The cells were then exposed to goat anti‐megalin IgG (1:100) with Can Get Signal^® solution B at 4°C overnight. The primary antibody was detected with Alexa Fluor 633‐conjugated donkey anti‐goat IgG. For nuclear staining, cells were incubated with DAPI at room temperature for 5 min. The cells were observed with a confocal fluorescence microscope (FV1000; Olympus, Tokyo, Japan).
To discriminate G1 phase cells from G0 phase cells, immunocytochemistry for Cdt1, which is specifically expressed during G1 phase (Wohlschlegel et al. 2000 (link); Nishitani et al. 2001 (link); Xouri et al. 2004 (link), 2007 (link); Sakaue‐Sawano et al. 2008 (link)), was also performed. After PT and DT cells were permeabilized with 0.5% Triton X‐100, the cells were incubated with rabbit anti‐Cdt1 IgG (1:100) and then with Alexa Fluor 546‐conjugated goat anti‐rabbit IgG.