Eclipse e600

Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E) and CD68 antibody at the Memorial Medical Center Histology Laboratory, Springfield IL. Quantification of adipocyte size was performed on H&E-stained sections using Image J software (version1.48e) Liver, VAT, and SAT were fixed in PBS + 10% formalin for 24 h, then stored in 70% ethanol. Single-blind, randomized images were obtained using a Nikon Eclipse E-600 microscopeequipped with an Olympus-750 video camera system. Adipocytes’ size was quantified using Image J Software (version1.48e) by an experimenter blinded to experimental conditions.

Samples were fixed in 4% paraformaldehyde and embedded in paraffin before being cut and mounted on polysine glass slides. Those sections were deparaffinized and rehydrated using standard laboratory procedures, then post fixed in 4% paraformaldehyde in phosphate buffered saline for 10 minutes. Heat mediated antigen retrieval was carried out for 20 min in 0.05% citraconic anhydride buffer pH 7.4 for all stainings except for BMP-1 where 10 mM citrate buffer pH 6.0 was used. Then, primary antibodies in 1.25% bovine serum albumin (BSA) were applied (according to Table 2) and incubated overnight at 4°C. Negative controls were made omitting primary antibody, while for ADAMTS-2, ADAMTS-3 and BMP-1, isotype controls were used. After rinsing, secondary antibodies in 12.5% BSA were applied at room temperature for 1.5 hours. The secondary antibodies used were goat anti-rabbit IgG conjugated to Alexa 488 (Invitrogen, Carsbad, CA) and donkey anti-mouse IgG conjugated to Cy3 (Jackson Immuno Research Europe, Newmarket, UK), used at 5.0 and 1.4 µg/ml, respectively. The stained sections were mounted with prolong gold antifade (Invitrogen), containing DAPI for nuclear stain. Imaging was done using an upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III camera.

Kidneys were collected at time of euthanasia and 1/3 of the spleen was preserved in 10% formalin for at least 24 hours, processed, paraffin embedded, and cut into 5 μm sections. After deparaffinizing and rehydrating, spleen sections were stained with anti-PAX5 (Abcam, ab140341 polyclonal). The ImmPRESS HRP reagent and ImmPACT DAB substrate were used to detect anti-PAX5 primary antibody as a brown pigment. Kidney sections were washed in distilled water, counter-stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) stain and dehydrated through an ethanol series. Slides were imaged on a Nikon Eclipse E600 supplied with an Olympus DP70 camera. Automated quantification was performed using a custom macro written for ImageJ software (NIH, imagej.nih.gov/ij/). All H&E and PAS slides were reviewed by a transplant pathologist (WZ) blinded to study groups and scored for rejection according to Banff 2013 guidelines.[23 ]

Kidneys were collected at time of euthanasia 1/3 of the spleen was preserved in 10% formalin for at least 24 hours, processed, paraffin embedded, cut into 5 μm sections. After deparaffinizing rehydrating, sections were stained with anti-PAX5 (Abcam, ab140341 polyclonal) or C4d (anti-C4d polyclonal antibody, American Research Products, Inc.) antibody overnight. The ImmPRESS HRP reagent ImmPACT DAB substrate were used to detect anti-PAX5 primary antibody as a brown pigment. Tissue sections were washed in distilled water, counter-stained with hematoxylin dehydrated through an ethanol series. Immunoperoxidase staining was done as previously described.[16 , 17 (link)] Slides were imaged on a Nikon Eclipse E600 supplied with an Olympus DP70 camera. Automated quantification was performed using a custom macro written for ImageJ software (NIH, imagej.nih.gov/ij/). All hematoxylin-eosin C4d slides were reviewed by a transplant pathologist scored for peritubular capillaritis (ptc), glomerulitis (g), tubulitis (t), vasculitis (v), interstitial inflammation (i), mi (microcirculation inflammation) C4d staining, according to Banff 2013. Antibody mediated rejection (ABMR) acute cellular mediated rejection (ACMR) scores were calculated according to Banff 2013 guidelines.[18 ]

The amount of pigment in the liver tissue was assessed after section staining according to the standard hematoxylin, eosin and saffron protocol: frozen sections were air dried to the slides, stained with Mayer’s Haemalaun solution (Merck Millipore), with alcoholic eosin Y (1%) and with alcoholic saffron (1%) and mounted in Coverquick 4000 (Labonord) mounting media. In order to quantify pigment content in the HES sections, 24 slides per treatment and time point were photographed under a Nikon Eclipse E600 microscope using an Olympus DP70 digital camera. Between 3 and 5 pictures were randomly taken along the slide, giving a total of 72 RBG pictures. Each picture has been imported into Ecognition Developer [version 8.7, Trimble Geospatial Imaging, Westminster, CO, USA]. We processed chessboard segmentation (1 pixel = 1 object) over the images. Then, using a 2-class typology (pigment or not), we manually defined a brightness threshold to discriminate between the two classes and classified the images according to this threshold. We rasterized the results of the classifications and estimated the proportion of each class.