Vimentin

Paraffin-embedded sections (3 μm) of renal cortical samples were examined under blinded conditions by a pathologist and a nephrologist. As described previously, the level of tubulo-interstitial fibrosis were investigated using Sirius red staining [24 (link)] and tissue remodeling by immunohistochemical assessment of vimentin expression (1/500, Cell Marque, Rocklin, CA, USA).
Frozen cortex sections (5 μm) were used to investigate LOX-1 and TGFβ expression by double immunofluorescence localization. We used a rabbit primary antibody at 1/100 (Abcam, Paris, France) and a goat anti-rabbit secondary antibody coupled to Alexa 488 fluorochrome (1/1000, Life Technologies, Saint Aubin, France) for LOX-1 expression and a mouse primary antibody at 1/100 (Santa Cruz, CA, USA) and a goat anti-mouse secondary antibody coupled to Alexa 568 Fluorochrome (1/1000, Life Technologies) for TGFβ.

The paraffin blocks were semiserially sectioned at 5 μm intervals and mounted on slides and processed for immunohistochemical staining. Standard conditions included immunostaining of three separate groups subjected to the same experimental conditions: (i) a group containing the accreta placentas and age-matched normal placentas (36 gw), (ii) a group containing increta and percreta placentas and the age-matched controls (38 gw), and (iii) a group comprising healthy placentas from 36 and 38 gw. The primary antibodies were rabbit polyclonal IgGs against human CRIPTO-1 protein (Santa Cruz Biotechnology, Santa Cruz, CA), cytokeratin LMW, and vimentin (Cell Marque Corporation, CA, EUA), respectively, diluted at 1 : 100, 1 : 350, and 1 : 100. Goat anti-rabbit and goat anti-mouse IgG (KPL, Kirkegaard & Perry Laboratories, Inc, USA) were used as second antibodies at 1 : 100 dilutions. The antigens in the sections were visualized using a DAB substrate kit for peroxidase (Vector Laboratories Inc., CA). Slides were counterstained with Mayer's hematoxylin. Sections from each placental group were used as negative controls with the primary antibody replaced with Tris-buffered saline or nonimmune rabbit serum.

KCI-MENG1 cells or KCI-MENG1-LPSX cells (dissociated cells from second generation xenograft mouse tumor) were seeded onto Millicell^® EZ slides (EMD Millipore, Billerica, MA, USA) and fixed with 4% paraformaldehyde before proceeding with immunostaining procedures using either the mouse or rabbit VECTASTAIN^® Elite ABC kit (Vector Labs) following the manufacturer’s protocol. Primary antibodies used targeted the following proteins: EMA (cat.#247M-94), PR (cat.#323R-14), Ki-67 (cat.#275R-14), vimentin (cat.#347R-14; all from CellMarque, Rocklin, CA, USA), and N-cadherin (cat.#NBP1-48309, Novus Biologicals, Littleton, CO, USA). All primary antibodies were used at a 1:100 dilution. The peroxidase substrate used was Vector ImmPACT^® DAB solution (cat.#SK-4105, Vector Labs) and sections were mounted with VectaMount™ (cat.#H-500, Vector Labs).