Axio scan z1 scanner

The sections were stained with hematoxylin-eosin (HE) and Picrosirius Red for histological evaluation and fixed in 10% paraformaldehyde, and embedded in paraffin. Then, 5-μm-thick sections were cut from the blocks, affixed to glass slides coated with poly-L-lysine, examined under a microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan), and evaluated with a digital image analysis system. Immunohistochemically analysis was performed as previously described by Jones et al. 2008 [18 (link)]. Briefly, the skin samples were incubated with a primary antibody applied for 1 h at room temperature. Tissue sections were then incubated with TGF-β primary antibody (1:200, Abcam Inc; http://www.abcam.com, accessed on 28 February 2023) for 1 h at 25 °C and incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at 25 °C, followed by washing and visualized using an isothiocyanate-conjugated secondary antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and imaged with an optical microscope Axio Scan.Z1 scanner (Carl Zeiss, Germany).

Immunohistochemistry (IHC) was performed on 3-μm formalin-fixed, paraffin-embedded sections from the BM. The following primary antibodies were used: anti- keratinocyte-derived cytokine (KC) (Cloud-Clone Corp., Houston, TX, USA; catalog no. PAA041Mu01; 1:50), anti-macrophage inflammatory protein 2 (MIP-2) (Cloud-Clone Corp.; catalog no. PAB603Mu01; 1:100), anti-lipopolysaccharide-inducible CXC chemokine (LIX) (Cloud-Clone Corp.; catalog no. PAA860Mu01; 1:100), anti-MMP2 (Abcam; catalog no. ab37150; 1:200), anti-MMP9 (Abcam; catalog no. ab38898; 1:200), and anti-SDF-1 (Abcam; catalog no. ab9797; 1:500). In the case of anti-KC, anti-MIP-2, anti-LIX, anti-MMP9, and anti-SDF-1, antigen retrieval was performed using a citrate buffer. All slides were scanned using a virtual microscopy scanner (Axio Scan Z1 scanner; Carl Zeiss, Jena, Germany); positive contributions were calculated by summing the highly positive, positive, and low-positive fractions for each staining using the IHC profiler Plugin of ImageJ [20 (link)].

After double immunofluorescence, the number of HA expressing motor neurons (detected by anti-HA antibody) which do or do not present mitochondrial sparse distribution (detected using anti-cytochrome C antibody) were counted for each mouse in a blind manner on every 8th section of the lumbar spinal cord (70 to 130 HA-positive motor neurons were recorded for each mouse). For quantification of HA signal intensity in motor neurons, sections were stained for the HA epitope tag. After staining, images of the spinal cord were acquired using Axio Scan Z1 scanner (Zeiss, Oberkochen, Germany). The motor neurons in the ventral horn of the spinal cord were measured for their fluorescence intensity using Fuji software. Measures were realized from every 8th 20 μm sciatic nerve section, corresponding to a total of 5–7 sections per animal. The ventral horn was circled, a common detection threshold was set up for every section and the average fluorescence intensity was calculated. For quantification of RAB9 and CytC immunofluorescence signal intensity, triple immunofluorescence stainings were performed using anti-RAB9A (detected in far red), anti-CYCS (detected in red) and anti-GFP (detected in green). Each Cell expressing GFP was circled, intensity of red and far red was recorded. A total number of 20 to 70 transfected cells were recorded for each plasmid construct.

Longitudinal heart and lung sections (4 µm) were obtained from treated mice (control and fibrotic). Organs were fixed in 4% paraformaldehyde, followed by gradient alcohol dehydration and paraffin embedding. COL fibers were stained with Masson's trichrome stain as well as Picrosirious red stain. Masson's trichrome staining was performed using Harrys' hematoxylin (Merk ref.:1.09253) and staining with Fuchina Ponceau (Acid fuchsine, merk ref.: 1.05231; and Xylidine Ponceau, sigma ref.: P2395) was used as contrast stain. Phosphomolybdic acid (Pancrea ref.: 1310031.1608) was used to label the collagen with light green (Merck ref.: 1.15941).
Picrosirious staining was performed by incubating Harrys' hematoxylin (Merk ref.:1.09253) as a contrast stain followed by Picrosirious red solution (SctTek ref.: SRS500) to stain the collagens. Complete scanning of all sections was performed in bright field with the Zeiss Axioscan Z1 scanner using the ×10/0.45 Plan Apo objective. Images were exported as JPG files with a pixel size of 0.884 mm/ pixel and analyzed with ImageJ 1.52i software.

Histological slides were prepared with hematoxylin-eosin (HE) stain and were used to mark areas of interest for performing tissue sample array arrangement or Tissue Microarray (TMA). Next, two 4 µm-thick paraffin-embedded sections of the TMA blocks were transferred to electrically charged Star Frost™ (Braunschweig, Germany) slides and incubated with an anti-Desmine (ab8592; Abcam, Cambridge, UK), anti-connexin 43 (ab217676; Abcam, Cambridge, UK) and anti-factor VIII (275376; Abcam, Cambridge, UK) overnight in a humidified chamber at a temperature between 2–8 °C [28 (link)].
Immunoreactivity was developed by adding DAB chromogen-substrate solution (Spring) to the slides. Harris hematoxylin was used for counterstaining. Positive and negative controls were performed in parallel with all reactions. The slides were scanned using the Axio Scan.Z1 scanner (Carl Zeiss, Jena, Germany). The generated files were fragmented into single images and approximately 25 images were selected for analysis. The areas of immunopositive markings for the antiprotein antibodies were quantified using Image-Pro Plus version 4.5 software (Media Cybernetics, Rockville, MD, USA). The immunopositive objects were selected using a “mask” to standardize and automate the process. Numerical data of the immunopositive marking area were generated and subsequently exported to an Excel spreadsheet.

FFPE samples were obtained and fixed, after that, they underwent staining with hematoxylin and eosin-H&E (Harris Hematoxylin: NewProv, Cod. PA203, Pinhais, Brazil; Eosin: BIOTEC Reagentes Analíticos, Cod. 4371, Pinhais, Brazil). Subsequently, specific staining for TLR4; ACE2; IL-1β; IL-18; NF-κB; ASC; NLRP-3 (or NALP); CASP1; CASP9; GDSM-D; NOX4; TNF-α was performed, and the slides were then scanned using Axio Scan.Z1 Scanner (ZEISS, Jena, Germany), and then ZEN Blue Edition (ZEISS, Jena, Germany) was utilized to randomly generate high-power fields (HPF  =  40× objective). Images were randomly generated by the software, with no investigator’s interference. The immunopositivity areas were measured by the Image-Pro Plus software version 4.5 (Media Cybernetics, Rockville, MD, USA). Subsequently, these stained areas were converted into percentages per total tissue area to enable statistical analysis. This study analyzed overall marker expression. Staining information can be found on supplementary Table S1

Footpad lesions and 5 mm long tail pieces were fixed in 4% PFA. Tail pieces were also decalcified with Shandon TBD-1 Decalcifier (Thermofisher Scientific) for 24 hrs at 4°C. All samples were embedded into paraffin and 5 μm transversal sections were obtained. Sections were stained with Masson’s Trichrome Blue by the Mouse Pathology Facility at the University of Lausanne. Entire sample area was acquired on AxioScan.Z1 scanner (Zeiss) with Plan Apochromat 20x/0.80 objective using Brightfield contrast or NanoZoomer S60 (Hamamatsu Photonics K.K.) scanner with Nikon Plan Apochromat 20x/0.75 objective using Brightfield contrast. Images were obtained using the following software: Zen Blue (Zeiss) for AxioScan.Z1 scanner or NPD.scan3.3 (Hamamatsu Photonics K.K.) for NanoZoomer S60 scanner. Representative images of the sections are shown.