Scn400 slide scanner

Following microCT scanning, femurs were analyzed for osteoclast abundance in the distal metaphyseal trabecular bone using histomorphometry, as done previously (Putnam et al., 2019 (link)). Femurs were decalcified in 20% EDTA for 3 days, paraffin embedded, and sectioned on a Leica RM2255 microtome (Leica Biosystems, Buffalo Grove, IL, USA) at a thickness of 4 μm. Sections were stained for tartrate-resistant acid phosphatase (TRAP) with hematoxylin counterstain. TRAP-stained slides were imaged with a Leica SCN400 Slide Scanner (Leica Biosystems) in brightfield at 20X or on a Cytation 5 Imaging System [BioTek Instruments, Winooski, VT, USA] in brightfield at 10X and analyzed with Bioquant software (Bioquant Image Analysis Corporation, Nashville, TN, USA). Bioquant software was used to measure osteoclast number, osteoclast surface, and bone surface in the trabeculae of the distal metaphysis of the femur proximal to the growth plate on the TRAP-stained slides. According to ASBMR standards (Dempster et al., 2013 (link)), osteoclast number, osteoclast surface, and bone surface were measured to calculate osteoclast number per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS).

Both normal tissue fragments and DTs were fixed in 4% paraformaldehyde (VWR, Leuven, Belgium, 9713 for 24 h at room temperature (RT)). They were alcohol-dehydrated, immersed in xylene and embedded in paraffin. The tissues were then cut into 5 µm-thick sections, followed by deparaffination and rehydration steps by means of toluene and alcohol baths. H and E staining was realized by immersion of slides in different baths in this order: tap water, distilled water, Mayer’s Hematoxylin (Sigma-Aldrich, MHS32), tap water, distilled water and in erythrosin B (VWR, 1.159.360.025). Masson’s trichrome was realized by immersion of slides in different baths in this order: tap water, distilled water, Mayer’s Hematoxylin, tap water, distilled water, ponceau (Sigma-Aldrich, P2395,)-fuchsin (VWR, 1052310025) solution, distilled water, phosphomolybdic acid (Sigma-Aldrich, 79560), distilled water and in methylene blue (Merck, Overijse, Belgium, 1163160050). Following staining procedures, slides were dehydrated and mounted using Dako mounting medium. General observation of slides were realized using a standard optical microscope (E200LED, Nikon, Amsterdam, The Netherlands). The slides were then scanned with the Leica SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany) and images were captured using Aperio Imagescope software (Leica Biosystems, Vista, CA, USA).

Formalin‐fixed paraffin‐embedded (FFPE) tissue sections from the paired tumor and paracancer tissue of ICC were dewaxed and hydrated with xylene and gradient alcohol, respectively. For lectin histochemistry, Cy5‐labeled WFA was applied to detect the LacdiNAc structures according to previously described protocols [35 (link)] with some modifications. Briefly, the sections were washed, blocked with 5% (w/v) BSA and 0.04% Triton for 1 h and incubated with 1 μg·μL⁻¹ Cy5‐labeled WFA overnight in the dark at RT, followed by staining with DAPI (1 μg·mL⁻¹ in PBS; Solarbio, Beijing, China). The fluorescence images were acquired by a fluorescence microscopy, and the relative intensity of fluorescence was quantified by imagej software (http://imagej.net).
For immunohistochemistry, the pretreated FFPE tissue sections were blocked and incubated with anti‐B4GALNT3 antibody (orb31745l Biorbyt, UK) or anti‐B4GALNT4 antibody (orb546266; Biorbyt, Cambridgeshire, UK) at a 1 : 100 dilution, followed by detection using the PV and DAB chromogenic kits (Servicebio, Wuhan, China). The slides were scanned using Leica SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany), and five different fields of view were randomly selected in each section for quantification.

We utilized the same IHC slide sets from our previous study.¹⁴ While in our previous study, we focused only on CD138+ plasma cells, a marker of endometrial inflammation, in the stroma to evaluate endometrial immune status in women with PCOS and in RIF patients, the present study extended our examination to include both epithelial and stromal cell compartments with the aim of investigating epithelial-to-stromal proportions along the entire menstrual cycle, as well as in the context of these two infertility-associated conditions. IHC staining of the endometrial biopsies from controls (n=73) and women with PCOS (n=91) was performed at the University of Oulu, Oulu, Finland. The tissue samples from RIF patients (n=29) were processed at Tartu University Hospital, Tartu, Estonia. All of the stainings were performed following standard hematoxylin and eosin staining and IHC protocols.^{14 ,18} The stained slides were digitalized using a Leica SCN 400 Slide Scanner (Leica, Biosystems) and uploaded to Aiforia Hub (Aiforia Technologies Oy, Helsinki, Finland) (Fig. 1).

Slides were imaged using a Nikon Eclipse E800 microscope with NIS-Elements Basic Research Software or by the Vanderbilt University Medical Center Digital Histology Shared Resource (DHSR). For chromogenic IHC, whole slides were imaged by the DHSR using a Leica SCN400 Slide Scanner (Leica Biosystems). For fluorescence staining, whole slides were imaged by the DHSR using an Aperio Versa 200 automated slide scanner (Leica Biosystems) and visualized using Aperio ImageScope (v12.4.3) (Leica Biosystems). Quantification was performed, according to a protocol blinded to genotype, by counting positive cells in well-oriented crypts or crypt-villus units.

To quantify the atherosclerotic lesions in the aortic root, upper part of heart was cut in the ascending aorta and the proximal sample containing the aortic sinus was embedded in Tissue Tek OCT (Optimal Cutting Temperature) compound (Miles Scientific, Naperville, IL, USA) and then stored at −80°C. Serial cryostat sections (6 µm) were prepared on a cryostat (Leica Microsystems, Buffalo, NY, USA). In brief, atherosclerotic lesions in the aortic root were examined at 6 locations and each separated by 90 µm, 9 serial sections were prepared from each location. These sections were stained with oil red O and counterstained with Mayer's hematoxylin. Whole slide images were produced with Leica SCN400 slide scanner (Leica Biosystems, Buffalo Grove, IL, USA), managed with the image server, Digital Image Hub (Leica Biosystems). In each case, average value for 3 serial sections at each of 6 locations of each animal was used for analysis. The lipid composition of the lesion was determined by calculating the percent of the oil red O positive area to the area of aorta root using the public domain software, ImageJ software. The area of aorta root was manually selected and the positive stained area within the aorta root was measured by adjusting the threshold value. The remaining sections were used for immunohistochemical analysis as described below.