Entellan new mounting medium

The lungs of the rats were analyzed by immunohistochemistry (IHC) using CD90 antibody (Abcam, ab133350). The formalin fixed paraffin-embedded (FFPE) lungs were sectioned into 3-5μm-thick sections using a microtome and mounted on polylysine-treated slides. The IHC assay was performed by a service facility. Heat-mediated antigen retrieval was performed at 97°C for 20 min, transferred to 65°C and washed in PBS. To block endogenous peroxidase, the tissues were incubated with hydrogen peroxide for 10min at room temperature, washed in in PBS for 5 min. After blocking of non-specific sites, the tissues were incubated for 40 min with polyclonal antibody against CD90 (1:200 dilution) at Dako Flex solution, and the slides were washed two times in PBS for 5 min. The slides were incubated with rabbit secondary antibody for 15 min and washed again. Then, tissues were incubated for 15min with the biotinylated link and with streptavidin conjugated to horseradish peroxidase for 20 min. Tissues were revealed using the DAB kit (Dako North America) and counterstained with Harris hematoxylin. The sections were washed in water, dehydrated in Ethanol for 30 seconds (70%, 95%, 100%), xylene embedded and assembled with Entellan® new mounting medium (Merck).

Fresh-frozen samples were initially rinsed with distilled water and subsequently stained for 2 min using Weigert's iron hematoxylin working solution. A thorough 10 min wash with running tap water followed. Then, tissue sections underwent a 15 s acid ethanol immersion (or 3 quick dips), followed by a 5 min tap water rinse. After that, a 5 min fast green staining was applied. This was succeeded by a brief 10-15 s 1% acetic acid treatment and a 10 min 0.01% Safranin-O staining. Sequential dehydration and clarification of sections were then performed using 70%, 90%, and 100% ethanol, followed by xylene. Subsequently, specimens were preserved with Entellan New Mounting Medium (Merck KGaA, Darmstadt, Germany), employing an adapted version of Kawamoto's film technique tailored for high-resolution microscopy 11 (link), 13 (link).

Sectioning, staining and microscopic observations were carried out as described by Tsuge et al. (1996) (link). Briefly, tissue samples were fixed with formalin-acetic acid-alcohol solution and dehydrated with ethanol series and embedded in Technovit 7100 resin (Kulzer and Co., Wehrheim, Germany), followed by 10-12 μm-thickness sectioning with a microtome (Microm HM360, Thermo Fischer Scientific, USA). Sections were stained with 0.1% (w/v) toluidine blue (Sigma) in 0.1 M phosphate buffer (pH 7.0), washed with water, dried, and mounted on glass slides with Entellan new mounting medium (EMD, Millipore, Burlington, MA, USA). Microphotography was done using a differential interference contrast microscope (DM4500, Leica, Germany).