Cellsens entry software

The tissue samples were fixed in 4% paraformaldehyde buffer for 24 h and then were embedded in paraffin. The paraffin-embedded tissues were cut into 5-μm-thick sections by a microtome. The xylene and ethanol (100, 100, 90, 80, and 70%) were used to deparaffinize and rehydrate. Then, the sections were stained in hematoxylin and eosin (H&E) and images were captured by a light microscope (BX51, Olympus, Tokyo, Japan). In each group, 10 mice were used and three sections with 30 HPFs were observed per mouse (i.e., 10 HPFs per section). The morphological parameters were measured using the CellSens Entry software (Olympus, Tokyo, Japan). The villus height was measured from the baseline to the tip of the highest one and the crypt depth was measured from the baseline to the bottom of the deepest one (Gallier et al., 2013 (link)).

A histopathological analysis was performed to characterize the cellular interaction between the wheat and Pst. BSMV-infected wheat leaves with Pst were sampled at 24, 48 and 120 hpi. Leaf segments cut from the inoculated leaves were fixed and decolorized with thanol/trichloromethane (3:1 v/v) containing 0.15% (w/v) trichloroacetic acid for 3–5 days. The segments were soaked in saturated chloral hydrate until translucent and then stained with wheat germ agglutinin (WGA) conjugated to Alexa-488 (Invitrogen)53 (link). The hyphal length, colony size and number of hyphal branches of stained tissues were examined under blue light excitation (excitation wavelength 450–480 nm and emission wavelength 515 nm) with an Olympus BX-53 microscope (Olympus Corporation, Japan) and calculated with the cellSens Entry software (Olympus Corporation, Japan) as described54 (link). H₂O₂ was stained in situ using 3,3-diaminobenzidine (DAB; Amresco, Solon, OH, USA)55 . The infection sites at which appressoria had formed over the stomata were considered to have successful penetration, and at least 50 infection sites were examined on each of five randomly selected leaf segments per treatment.

The fluorescently labeled aptamer-bead complex was analyzed using a CKX53 microscope (Olympus, Tokyo, Japan). After aptamer-bead coupling, a 5 μL aliquot of beads was transferred onto a slide glass and covered with the cover glass. The microscope was controlled by a computer with the CellSens Entry software (Olympus) at 200× magnification. Photographic images were captured in a completely darkened room, and the entire process was completed within 10 minutes.
The fluorescence intensity on the images was measured using ImageJ software (National Institutes of Health, MD, USA). First, Photoshop (Adobe Systems, CA, USA) was used to select regions of interest (ROIs), measuring 100 × 100 pixels from the images. ROIs of the same size were selected and cut with Photoshop in the same positions in all images to maintain a standard in the analysis of the measurements. Next, ImageJ software was used to extract the average brightness value from the ROI. Each pixel was assigned a numerical value that represented its brightness on the grayscale 22, ranging from 0 (black pixels) to 255 (white pixels).

After four weeks of tumor injection, the mice were euthanized and their brains were surgically removed and fixed in 10% neutral buffered formalin.
For HE staining, the 5 μm slides were deparaffinized and brought through a graded ethanol series to dH₂O before the nuclei were stained with hematoxylin; the sections were then rinsed in running tap water and stained with eosin before being dehydrated and mounted. Pictures were taken using an Olympus upright BX53 microscope (Olympus). CellSens Entry software equipped with a digital CCD camera (Olympus DP22) was used.
For IF staining, Paraffin-embedded sections containing mice brains were deparaffinized, rehydrated, and subjected to antigen retrieval for staining of the M1 macrophage marker iNOS (CST, 13120). The processed brain tumor sections were incubated with primary antibody anti-iNOS (1:500) overnight at 4°C. Then, the sections were washed with PBS and followed by incubation with FITC (492-520 nm) (ZSGB-BIO, ZF-0311, China) for staining at room temperature for 60 min. Excess antibody was washed out with TBS, sections were counterstained with DAPI at room temperature for 5 min. All pictures were taken using an Olympus upright BX53 microscope (Olympus).

As described in a previous study, the desmoplastic reaction status was evaluated according to the ratio of the area of the tumour-associated stroma to that of the whole tumour tissue [32 (link)]. Histologic images were obtained using the light microscope (Olympus BX53®, Tokyo, Japan) with an attached digital camera (DP27®, Japan) and Olympus cellSens™ Entry software. The subjected areas were measured on haematoxylin and eosin-stained OSCC tumour tissue sections with ImageJ software. The desmoplasia status was further divided into two groups according to the calculated ratio: high (ratio ≥ 1) and low desmoplastic (ratio < 1) reaction.