HER1 Signaling Mediates Extravillous Trophoblast Differentiation in Humans: Fluorescent Immunohistochemistry

Fluorescent Immunohistochemistry
Placental villous explants were fixed and processed to paraffin blocks from which 5-im sections were cut and adhered to Superfrost Plus glass slides (VWR, Mississauga, ON). Sections were deparaffinized in xylene and rehydrated through a descending concentration gradient of ethanol. Antigen retrieval was performed using either microwave pretreatment in 10 mM sodium citrate buffer (pH 6; Sigma), 0.02% Triton X100 (Sigma), 0.125% Trypsin (Sigma), or 10 ig/ml proteinase K (Roche, Montreal, QC, Canada) at 37°C (Table 1). Slides used for immunofluorescence detection were rapidly exposed to 0.1% Sudan Black in 70% ethanol to prevent autofluorescence (Sigma) and washed prior to blocking for nonspecific binding in serum-free DAKO protein block (DAKO, Mississauga, ON, Canada) for 1 h. All primary and secondary antibodies used in these procedures are detailed in Table 1. Primary antibodies were prepared in DakoCytomation Antibody Diluent with Background-Reducing Components (DAKO) and were incubated on sections overnight at 48C. In control experiments, primary antibodies were replaced with DAKO blocking solution or mouse or rabbit IgG at the same concentration as the primary antibody. Secondary biotinylated antibodies were prepared in PBS +
0.04% Azide (Sigma) + 0.008% gelatin (Sigma), used at 1:300, and detected using Streptavidin-Alexa488 (1:1000; Invitrogen); cells were counterstained with the nuclear stain Hoescht 33258 (1 lg/ml, 1 h; Sigma). All incubations were performed in a light-protected incubation chamber. Slides were washed in PBS and mounted in 50% glycerol/50% PBS for deconvolution microscopy. Immunofluorescent images were captured using a Sony Interline ICX285ER Progressive scan camera and an Olympus IX70 microscope (Olympus America Inc., Melville, NY). Images were collected using Resolve3D Image acquisition software and deconvolved using Deltavision softWoRx 2.50 software (Applied Precision, Issaquah, WA).

Maintenance of Cell Lines
The human placental choriocarcinoma cell line JAR was obtained from American Type Culture Collection (Manassas, VA). Cells were maintained at 37°C under atmospheric O2 plus 5% CO2 in 10% FBS RPMI, supplemented with 100 ig/ml amphotericin B, 100 ig/ml streptomycin, and 100 U/ml penicillin. Prior to treatment, JAR cells were serum-starved for 24 h in 0.2% BSA RPMI media supplemented with Normocin (Cedarlane) serum free media (SFM).

TABLE 1. Antibodies, dilutions, and antigen retrieval for placental villous explant immunohistochemistry.

Antibody Host Dilution Source Unmaskinga
Cx40 Rabbit 1: 125 Invitrogen Triton 0.02%
Mouse 1: 100 Abcam Triton 0.02%
a1 integrin Rabbit 1: 500 Cedarlane Proteinase K (10 ig/ml)
HER1 Mouse 1: 250 Santa Cruz (CA) Trypsin 0.125%
HER2 Rabbit 1: 300 DAKO 10 mM Sodium citrate pH 6, heat
Cy-3 anti-mouse 1: 200 Jackson Laboratories NA
FITC anti-rabbit 1: 200 Jackson Laboratories NA
Biotinylated anti-mouse 1: 300 DAKO NA
Biotinylated anti-rabbit 1: 300 DAKO NA
Alexa 488-conjugated streptavidin 1: 1000 Invitrogen NA


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