Rm2135

The operated lateral branch of the harvested DDFT was transected at the tendon bifurcation and fixed in 10% formalin for 7 days. Tendon samples were then processed using a Leica ASP300S tissue processor and embedded in paraffin wax. Using a rotary RM-2135 microtome (Leica Microsystems Ltd, Milton Keynes, UK), 5–7 μm sections were cut and placed onto Surgipath X-tra Adhesive glass slides (Leica Microsystems Ltd, Milton Keynes, UK) and stained with haematoxylin and eosin (H&E). High magnification images (100X) were taken using a Zeiss AX10 inverted microscope with an AxioCam HRc camera and Axiovision software (Zeiss, Cambridge, UK) in bright field mode from 3 distinct areas or zones in the tissue sections, namely the ES, MF and NT (normal tendon) zones. ES zones refer to tissue areas found in close proximity to fragments of the ES suture. MF zones relate to tissue areas lying immediately adjacent to the MF suture material (often removed during sectioning). NT zones refer to sites distant to the suture implants (ES and MF) and served as an internal control group.
Taking 6 random images per field, per sample, we used histomorphometric features to quantify immune cell (IC) count, fibroblast cell (FC) count, and foreign body giant cell (FBGC) count. Vascularity was evaluated semi-objectively using the following grading system:

Buds from the time points 0, 475 and 770 CU were fixed in 4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer (pH 7.2) for 16 h at 4°C and embedded in Paraplast Plus (Sigma‐Aldrich). Sections (7–10 μm) were cut using a microtome (RM2135; Leica Microsystems, Heidelberg, Germany) and collected on xylene‐coated slides (Superfrost Plus™ Adhesion Microscope Slides; Thermo Fisher Scientific, Gerhard Menzel BV & Co. KG, Braunschweig, Germany). For the cytological observation, slides were deparaffinized using xylene (Sigma‐Aldrich) and stained with 0.1% toluidine blue, and then air‐dried and mounted with DPX mounting medium (Honeywell Fluka, Charlotte, NC, USA). Callose deposition was observed using 0.1% of aniline blue. An in situ hybridization experiment was performed to localize the DAM4 expression domains and was conducted as previously described (Varotto et al., 2003 (link)). A detailed explanation of the protocol is reported in Methods S1 and Table S1.

Lung tissue samples were first fixed in a 10% neutral buffer formaldehyde solution for light microscopic examination. After fixation, tissue samples were placed in cassettes and washed under running water for 2 h. To remove water, tissues were passed through a series of increasing degrees of alcohol (60%, 70%, 80%, 90%, 96%, and 100%). The tissues were then passed through xylol for transparency and then embedded in paraffin. Sections were taken from the paraffin-embedded tissues with a rotary microtome (RM 2135, Leica Instruments, Nussloch, Germany).

HE and Nissl staining have been performed as previously reported [41 (link)]. Briefly, immediately after tDCS treatment on POD 3, the rats were anesthetized (1% pentobarbital sodium, 60 mg/kg i.p.) and transcardially perfused with 200 ml of 5 mM sodium phosphate-buffered 0.9% (w/v) saline (PBS, pH 7.2–7.4) followed by 500 ml of 4% paraformaldehyde in phosphate buffer (n = 6 for each group), then the brains were rapidly removed. After being dehydrated in ethanol and defatted in xylene, the brain was embedded in paraffin and 4 μm thick horizontal slices were obtained by using a rotary microtome (Leica RM2135, Leica Biosystems, Germany). For HE staining, the sections were dipped in hematoxylin for 3 min, washed in running tap water and destained in hydrochloric acid alcohol for several seconds. The sections were washed again and then dipped in eosin for 15 s. They were subsequently dehydrated in an alcohol gradient, cleared in xylene, and coverslipped. For Nissl staining, the sections were dipped in 1% toluidine blue for 30 min at 45 °C, and differentiated in 75% alcohol for seconds, then rinsed quickly in distilled water. Digital images were acquired using an inverted microscope (DMI4000B, Leica, Leica Biosystems, Germany).

Tissues from the brain (perilesional area [penumbra] located in the right hemisphere of the ischemic lesion), left lung, right kidney, and heart were extracted, fixed in 4% paraformaldehyde for 24 h, and embedded in paraffin. Tissue section (5 μm thick) were cut (Leica RM2135 Leica Biosystem, São Paulo, Brazil) and stained with hematoxylin and eosin. Eight fields of view per section were studied through a light microscope (Olympus BX51; Olympus Latin America, Brazil), at × 25, × 100, × 400 and × 1000 magnification. Perilesional brain, lung, kidney, and heart injury scores were quantified using a scale to represent the severity of apoptosis, edema, inflammation, and necrosis (0, no effect; 4, maximum severity). The extension of each feature was graded as 0 for no appearance and 4 for complete involvement. Final scores were calculated as the product of severity and extent of each feature, ranging from 0 to 16. The cumulated brain, lung, kidney, and heart injury score ranged from 0 to 64 [25 (link)]. All histological analyses were performed by two investigators (G.R. and V.L.C.), blinded to the group allocation. The scores of each expert were combined to yield a final score by arithmetic averaging [25 (link)]. The value of kappa was 0.86.

On the 30th day, 20 mice were anaesthetized as previously described and fixed via cardiac perfusion with 4% paraformaldehyde after flushing out the red blood cells with PBS. The whole brains were isolated and fixed in 4% paraformaldehyde for 1 day. The brains were then dehydrated in ethanol, defatted in xylene, embedded in paraffin, and serially sectioned at 5 μm thicknesses by using a rotary microtome (Leica RM2135, Leica Biosystems, Heidelberg, Germany). Subsequently, the sections were stained with HE according to procedure which has been reported [18 ]. The digital photograph of hippocampal CA1, CA3 and amygdala were observed by Leica DMI4000B (Leica Biosystems, Heidelberg, Germany) and a Hamamatsu Nano Zoomer scan SQ1.0 (Hamamatsu Photonics, Shizuoka, Japan) with NDP, respectively.