Dm2000 upright microscope

The rat lung was fixed in 10% formalin (Sigma) for 24 h, followed by embedment and 6-µm sectioning for hematoxylin & eosin (H&E) staining. Samples were dehydrated using graded ethanol, embedded in paraffin wax, and cut into sections using a Leica rotary microtome (thickness, 6 µm). Sections were deparaffinized with xylene, stained with Gills hematoxylin, and washed. Sections were then subsequently counterstained with 1% eosin, dehydrated with ethanol, and cleared with Neo-Clear (Darmstadt, Germany) before mounting using HistoMount (Atlanta, GA, USA). Sections were analyzed using a Leica DM2000 upright microscope (Wetzlar, Germany). The entire H&E-stained section was evaluated at low magnification (5× objective) for inflammatory cell infiltration. A 4-point scoring scale of cell infiltration was used to determine the grade of lung inflammation: 0 = normal; 1 = mild; 2 = intermediate; 3 = severe (19 (link)). Features examined were inflammatory cell infiltration, pulmonary congestion, and thickening of the alveolar septa. A total of 10 fields were evaluated randomly for each sample. The score for each group was the average score for all samples in the group. Quantitative analysis was performed in a blinded way.

The samples of DMASM-encapsulated 4.5-µm MIL-96 single particles or MIL-96-2 films was then mounted on the stage and illuminated using linearly polarized light. The filtered excitation light (470 ± 25 nm) from a LED light source (pE-300lite) was first allowed to pass a polarizer before hitting on the sample. The polarizer can be rotated with a constant speed to alter the direction of light polarization from 0° to 360°. The fluorescence signal passing through an emission filter (525 ± 25 nm) was captured by CCD detector. The experiment is done on a Leica DM2000 upright microscope.

Paraffin-embedded tissue sections (4 μm) were dewaxed in xylenes 3 times, passed through a series of graded alcohols, and washed in deionized water. For antigen retrieval, 1X CitraPlus AR Buffer (Biogenex) was pre-heated for 20 min in an Oster steamer before adding the slides for an additional 20 min. After cooling for 10 min, slides were washed 3 times in deionized water. Sequential blocking steps were performed—10 min in Bloxall (Vector Laboratories), 30 min in F_c Receptor Block (Biogenex) and 30 min in 1% normal horse serum (Vector Laboratories). Sections were incubated overnight at 4 °C in 1:200 rabbit IgG anti-CDKN2A/p16INK4a antibody (Abcam, ab108349). After washing 3 times in TBST, sections were incubated for 30 min in Vector ImmPRESS HRP Horse Anti-Rabbit IgG Polymer reagent. After washes, slides were developed for 3 min with Vector ImmPACT DAB. Sections were then counterstained with hematoxylin, dehydrated, cleared and mounted using VectaMount (Vector Laboratories). Images were captured using a Leica DM 2000 upright microscope.