Dm750 led biological microscope

The formalin-fixed lung tissues were embedded in paraffin, which were de-paraffinized using xylene and ethanol. For histological analysis, the de-paraffinized sections were stained with hematoxylin and eosin (H&E). For IHC analysis, antigen retrieval was conducted by boiling the sections in an antigen retrieval buffer (Abcam Cat No. ab93678, Cambridge, UK) for 20 min. Expression of specific proteins in lung tissue was determined by IHC using a Vectastain Elite ABC universal plus kit (PK-8200) according to manufacturer’s protocol. In brief, sections were incubated with the bloxall endogenous enzyme blocking solution for 10 min to quench endogenous peroxidase activity, followed by rinsing three times (5 min each) in TBST buffer (0.05% Tween-20). Blocking solution was applied for 20 min before sections were incubated with primary antibodies overnight at 4 °C in a humid chamber: anti-COX-2 (Cell Signaling, 1:200, Danvers, MA, USA), anti-Ki-67 (Cell Signaling, 1:400, Danvers, MA, USA), anti-caspase-3 (Cell Signaling, 1:1000, Danvers, MA, USA) and anti-GADD45A (Abcam 1:500, Cambridge, UK). Ki-67 was scored by counting the positively stained cells in 7 to 12 fields at various locations per group on mouse lung tissue (n = 5 mice). Lung sections were examined and imaged using a Leica DM750 LED biological microscope.

Histological procedures followed ref. ⁶³ . Specifically, primary branches were collected from the central third of panicles 12 and 16 d after heading and fixed in FAA (37% formaldehyde: ethanol: H₂O: acetic acid = 10:50:35:5), followed by a dehydration series in 50%, 70%, 85%, 95%, 100%, 100% and 100% ethanol and 25%, 50%, 75%, 100%, 100% and 100% Histo-Clear (National Diagnostics) series with ethanol as solvent. Paraplast (Leica Biosystems) was then added to each vial of samples and kept overnight, heated at 42 °C and placed in a 60 °C oven. The solution was replaced with molten Paraplast twice a day for 3 d. Samples were then embedded in paraffin using a Leica EG1150 tissue embedder, sectioned in 10-µm serial slices with a Leica RM2255 automated microtome and mounted on microscope slides at 42 °C on a Premiere XH-2001 Slide Warmer. Sections were then deparaffinized, rehydrated, stained with 0.05% (wt/vol) toluidine blue O for 1.5 min and then rinsed with water, dehydrated in ethanol, cleared with xylene and mounted with Permount Mounting Medium (Electron Microscopy Sciences). Images were taken using a Leica DM750 LED Biological Microscope with ICC50 camera module and Leica Acquire version 2.0 software. Experiments were repeated on three independent plants of each genotype.