Citrisolv

Slides were stained with hematoxylin and eosin (H&E) and by immunofluorescence (IF). For IF, sections (7 μm) were deparaffinized in CitriSolv (Fisher Scientific; Fairlawn, NJ, USA) and rehydrated through a series of graded ethanol to distilled water steps. Antigen retrieval (10 mM sodium citrate, pH 6.0, Sigma-Aldrich; St. Louis, Missouri, USA) and blocking (1:500 dilution; 100 μL goat serum: 50 mL TRIS-buffered saline, 0.1% Tween 20 (TBST) for 1 h), steps were performed prior to going through sequential wash (TBST) and the application of primary antibody. Primary antibodies conjugated to fluorophores included: CD14-FITC (Macrophage), CD68-PE (Macrophage), CD90-Alexa488 (MPC) or CD271-Alexa568 (MPC) (all BD Biosciences; Franklin Lakes, NJ, USA), and all slides were counterstained with the nucleic acid stain DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich; St. Louis, MS, USA) and mounted using FluorSave reagent (Calbiochem; Darmstadt, Germany). Isotype controls for fluorescein isothiocyanate (FITC) and phycoerythrin (PE) demonstrated little to no reactivity (Figure S5).
Imaging: Slides were imaged using a Plan-Apochromat objective (20×/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss; Oberkochen, Germany); DAPI (excitation 353 nm, emission 465 nm), Alexa488 (excitation 493 nm, emission 517 nm), and Alexa568 (excitation 565 nm, emission 576 nm).

Whole mount ISH was performed, essentially, as previously described [69 ] except BM purple (Roche) was used as the chromogenic substrate. Briefly, plasmids encoding cDNA for mouse Stat3, Scx, MyoG, or Sox9 were linearized and DIG-labeled complimentary probes were generated using a DIG-labeling kit (Roche). As indicated, probes were hybridized overnight and detected using alkaline phosphatase-labeled anti-DIG antibody with subsequent chromogenic substrate development. For sectioning, embryos stained as above were embedded into paraffin wax after clearing in CitriSolv (Fisher Scientific) and sectioned serially at 16μM by microtome.

Picrosirius red staining was done similarly to other studies [14 (link), 15 (link), 38 , 39 (link)]. Before sectioning, fixed muscles were embedded in 4% agarose. 200 μm thick longitudinal sections were sliced using Leica VT1000S. Sections were washed, dried for 60 min, and stained for 60 min in 0.1% (weight/volume) Direct Red 80 (Fisher) dissolved in saturated aqueous picric acid (Fisher). Sections were washed twice for 60 s in 0.5% acetic acid, then dehydrated with three 60 s washes of 100% ethanol. The sections were then cleared using CitriSolv (Fisher Scientific) for 3 min, and blotted with Permount (Fisher Scientific).
Full longitudinal sections were imaged via a 20X objective with brightfield illumination on a Leica DMi8 microscope and DFC9000GTC camera. Linearly polarized light imaging required a rotating polarizer in the beam path before and after the sample. A sequence of ten tiling scans were imaged at angles from 0 to 90° in increments of 10°. ECM architecture was quantified using a custom MATLAB script providing MicroECM alignment and MacroECM deviation parameters as described previously [14 (link)] in conjunction with the polarized light images.

Ovaries from 40-day-old CD1 mice were fixed in Modified Davidson's Fixative (Electron Microscopy Sciences, 64133-50) and paraffin-embedded. Five micron sections were deparaffinized in Citrisolv (Fisher, 04-355-121) and antigen retrieval achieved using Reveal Decloaker (Biocare Medical, RV1000). Sections were prepared for immunohistochemistry using Avidin/Biotin blocking kit (Vector Labs, SP-2001) in 10% normal goat serum in TBS. Sections were then incubated with diluted 1:100 HMGB1 primary antibody (Abcam, ab18256, lot GR135551-1) in 10% normal goat serum in TBS overnight at 4 °C. As a control for expression, the HMGB1 blocking peptide (Abcam, ab18650) was also used. A ratio of 1:5 HMGB1 antibody to HMGB1 peptide was incubated overnight at 4 °C prior to application. Sections were washed in TBS+0.1% Tween-20 and then incubated with diluted 1:200 biotinylated goat anti-rabbit secondary antibody from the Vectastain Elite ABC kit (Vector Labs, PK-6105) for 2 h at room temperature, washed again and followed by a 30 min incubation in the ABC reagent (Vector Labs, Vectastain Elite ABC kit) and a final wash. Binding was detected with diaminobenzidine (DAB; Vector Labs, SK-4100) for 6 min. Counterstaining was achieved using standard haematoxylin staining. All images were acquired and processed on an Evos fl auto inverted microscope using Evos software (Life Technologies).

For histological analysis, explant IVDs were fixed with 10% neutral buffered formalin for 24 hours, embedded in paraffin blocks, and 5 µm sections were prepared. Slides were de-paraffinized in Citrisolv (Fisher; Pittsburgh, PA), rehydrated through ethanol series and stained with hematoxylin and eosin or alcian blue.

Formalin-fixed placental tissue from normal or preeclamptic pregnancies were sectioned at 10 μm and deparaffinized with Citrisolv (Fisher Scientific, USA). Antigen retrieval was performed by heating the sections in 0.1 M sodium citrate (pH 6.0). Sections were blocked in blocking buffer (3% bovine serum albumin and 0.1% Triton X-100 in PBS) and then incubated with transthyretin polyclonal rabbit antibody (DAKO) overnight at 4 °C. Primary antibody-bound target proteins were visualized with goat anti-rabbit Alexa-Fluor 488 (Invitrogen). The specificity of transthyretin antibody was confirmed by blocking transthyretin staining by incubating the primary antibody with the immunogenic peptide or by using normal rabbit serum IgG instead of primary antibody. Images were processed with brightness/contrast adjustment using Photoshop CS2 (Adobe) at the same levels.