Lide110 scanner

Samples were diluted at a ratio of 1:5 and heated at 95 °C for 5 minutes. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. 5% milk was used to block the membranes in TBST for 1 hour at room temperature, incubated with primary antibodies at 4°C overnight, and incubated with horseradish peroxidase-conjugated secondary antibodies at 37 °C for 1 hour. Primary antibodies and dilutions were as follows: anti-TLR4 (1:1000; #ab13556, Abcam, Cambridge, UK), anti-Collagen I (1:1000; #ab138492, Abcam, Cambridge, UK), anti-OCN (1:1000; #ab133612, Abcam, Cambridge, UK), anti-Runx2 (1:1000; #ab236639, Abcam, Cambridge, UK), and anti-GAPDH (1:10000; #ab37168, Abcam, Cambridge, UK). Secondary antibodies were used to probe the blots, and protein was detected with a LiDE110 scanner (Canon, Tokyo, Japan).

PO activity in S. aureus-infected T. molitor larvae, pupae and adults injected with Neb-colloostatin or an analog solution or exposed to an ND-peptide solution (ND-Neb-colloostatin, ND-[Ala¹]-, [Val¹]-, [Hyp⁴]- or [Ach⁴]-colloostatin) was measured on the third day of the experiment according to a modified method described previously^{42 (link),43 (link)}. In brief, 1 μl of hemolymph was placed on white filter paper (Whatman No. 52) soaked in a 10-mM sodium phosphate buffer containing 2 mg/ml DL-DOPA (Sigma Aldrich). Subsequently, the filter papers with the samples were incubated for 30 min at room temperature and air-dried. The samples were then scanned with a Canon Lide 110 scanner (600 dpi, 8 bits, gray scale). Images have been transformed so that the darker samples correspond to the highest PO activity. Images of the samples were analyzed with ImageJ (ver. 2), measuring the color intensity of the samples in their central 40 × 40 pixel area. The PO activity assessment was based on the mean pixel value of each image.

Liver sections from mice of control and vaccinated groups and infected with 100 cercariae were collected 45 days post-infection to evaluate the effect of immunization in granuloma formation. The liver sections removed from the central part of the left lateral lobe were fixed with 10% buffered formaldehyde in PBS. Histological sections were performed using microtome (4 μm) and stained in a slide with Gomory's trichromic. The granulomas were counted in Axiolab Carl Zeiss microscope using 10× objective lens. All slides were digitized by the Canon Lide 110 scanner, in 300 dpi resolution. The pixels of each histological section were fully screened, with subsequent creation of a binary image and the total area of the cut was calculated. The area of the lower cutoff was used as a minimum standard of tissue to be statistically analyzed. The results were expressed by the number of granulomas per area of liver (mm²). The area of granulomas was obtained through the KS300 software contained in Carl Zeiss image analyzer. Fifteen granulomas from each mouse with a single well-defined egg were randomly chosen at a microscope with 20× objective lens and scanned through a Q-Color3 microcamera (Olympus). Using a digital pad, the total area of granulomas was measured and the results were expressed in square micrometers (μm²).

OCCM‐30 single‐layer cells were lysed with RIPA buffer (Beyotime) containing phosphatase inhibitors (Roche) and PMSF (Beyotime). Then, lysate was centrifuged, the supernatant was collected, and the total protein concentration was determined using the BCA Protein Assay Kit (Beyotime). Forty micrograms of total protein from each group diluted in loading buffer (Beyotime) was boiled at 95°C for 10 minutes, separated by electrophoresis in 10% SDS polyacrylamide gel (Servicebio) and wetly transferred to a PVDF membrane (Millipore). The membrane was subsequently blocked with 5% non‐fat milk for 1 hour. Afterwards, the membrane was incubated at 4°C overnight with the following primary antibodies: anti‐YAP (1:2000; CST), anti‐OCN (1:1000; Proteintech) and anti‐p‐p65 (S536; 1:1000; CST).
In the next day, the membrane was washed three times with TBST buffer, incubated with corresponding horseradish peroxidase‐conjugated secondary antibody for 1 hour at room temperature and then washed with TBST buffer three times. Blots were generated using the ECL reagent (Advansta) and detected using X‐ray films (Kodak). The protein bands were scanned using the LiDE 110 scanner (Canon) and quantified with densitometry analysis by using the ImageJ software (National Institutes of Health). Western blot analysis was repeated at least three times.