Dp74 cmos camera

Dried sections on charged glass were deparaffinized and rehydrated thorough xylene and graded alcohols into tap water. Rehydrated sections were submerged in a 0.1% solution of Picrosirius Red composed of Direct Red 80 (#365548; Sigma‐Aldrich) in a saturated aqueous solution of picric acid (#P6744; Sigma‐Aldrich) for 1 h at RT to achieve stain saturation. Slides were washed twice in acidified water (0.5% glacial acetic acid in distilled water) for 2 min each. Slides were dehydrated through an abbreviated 90% and absolute ethanol series, further dehydrated in xylene and mounted in CytoSeal XYL (Richard‐Allan/Thermo Fisher Scientific). Brightfield images were collected on an Olympus BX43 upright microscope equipped with an Olympus DP74 CMOS camera operated by cellSens Standard software (Olympus Corporation).

Captured sections were rehydrated and overnight epitope retrieval was performed in sodium citrate buffer at 60°C. Tissue sections were blocked with 5% BSA in TBS containing 0.2% Tween‐20 (TBST) at RT for 1 h and incubated overnight with primary antibodies for iNOS (Abcam; ab15323; rabbit polyclonal; 1:50), Arginase‐1 (Cell Signaling Technologies; #93668; rabbit monoclonal; 1:200), or Ly6G (Invitrogen; #14‐5931‐82; rat monoclonal; 1:100). Secondary antibodies were probed for 2 h at RT using HRP‐(rabbit) or AP‐(rat) conjugates (Jackson Immunolabs). Tissues were developed with ImmPACT DAB (HRP) or Vector Red (AP) substrate (Vector Labs). Arginase‐1 and Ly6G sections were counterstained with hematoxylin while iNOS sections were left without counterstain. Tissues were dehydrated through alcohol and xylene and mounted with Cytoseal XYL. Images were collected on an Olympus BX43 upright microscope equipped with an Olympus DP74 CMOS camera operated by cellSens Standard software. Images were quantified in ImageJ/FIJI.

The cuticle of the P. conspersa larvae was pierced under the saturated picric acid-based fixative with 3.6% formaldehyde and 2.3% copper acetate supplemented with mercuric chloride (Bouin-Hollande solution) (Levine et al., 1995 (link)). Following 1 h of fixation, the larvae were cut into three pieces and then fixed overnight at 4°C. After coarse washing in 70% ethanol, the tissue was dehydrated by an ethanol series (70%, 96%, and 100%, each twice for 20 min) and 100% chloroform (twice for 20 min), embedded in paraplast, and then sectioned to 10 μm.
For the labeling procedure, samples were deparaffinized in 100% xylene (twice for 10 min), rehydrated by subsequent incubation in 96% and 70% ethanol for 5 min each, and washed in distilled water for 5 min. The sections were treated with Lugol’s iodine followed by a 7.5% sodium thiosulfate solution to remove residual heavy metal ions and then washed in distilled water and stained with HT15 Trichrome Stain (Masson) Kit (Sigma-Aldrich, Burlington, United States) according to the manufacturer’s protocol. The stained sections were dehydrated and mounted in DPX mounting medium (Fluka, Buchs, Switzerland). High-resolution images were acquired using a BX63 microscope, DP74 CMOS camera, and cellSens software (Olympus, Tokyo, Japan) and by stitching multiple images together.