MK, CD105 and PTN Immunohistochemistry.
Protocol A was performed for fibroblast growth factor 2 (FGF2) the primary antibody was applied to the slide covering the placental specimen in different dilutions (1 in 100), (1 in 200), and (1 in 400) control, and a negative control IgG were optimised in the absence of citrate buffer. These slides in dilution 1 in 200 were incubated overnight at 2-8°C. However, in protocol B the procedure was performed in the presence of citrate buffer for the following heparin-binding growth factors VEGF, PLGF, FGF2, HGF, PDGF-BB, and CD105 except for HB-EGF, MK, and PTN, using placental specimens in different dilutions, control, and a negative control IgG were optimised.
The primary antibody was applied to the slide covering the placental specimen (n=87) in the presence of citrate buffer with different dilutions and optimised for VEGF primary antibody (goat, 1 in 50 dilution), HGF primary antibody (goat, 1 in 100 dilution) and PDGF-BB primary antibody (rabbit, 1 in 100 dilution) and control. After preparation of slides, the sections were deparaffinised by 2 changes of xylene, of 5 minutes each, then re-hydrated in 2 changes of absolute alcohol, for 3 minutes each. The re-hydrating procedure was continued by incubation with 90% alcohol for 3 minutes and then 70% alcohol for 3 minutes. The slide was blocked with (50% in PBS) diluted normal serum (horse serum) in PBS for 5 minutes and incubated with VEGF primary antibody (goat, 1 in 50 dilution); HGF primary antibody (goat, 1 in 100 dilution) and PDGF-BB primary antibody (rabbit, 1 in 100 dilution) for 30 minutes at room temperature. The sections were incubated in preheated citrate buffer with 1M Tris to at pH 8.0 with HCL, and 0.5M EDTA for 10 minutes in a microwave and boiled in a microwave for 4 minutes. The slides were left to cool in heated buffer for 20 minutes. Rinses and washes in PBS were carried out in between the following steps. The next steps of this protocol are described below.
The procedure was identical to that used for VEGF, HGF and PDGF-BB except after the sections were deparaffinised the slides for FGF2, PLGF, CD105, HB-EGF, IgG, MK and PTN were quenched by the endogenous peroxidase activity, and incubated with a blocking reagent 3% H2O2 in PBS for 1 minute. The slides were then washed in running tap water for 1 minute. The sections were incubated in preheated citrate buffer with 1M Tris to at pH 8.0 with HCL, and 0.5M EDTA, for 10 minutes in a microwave and boiled in a microwave for 4 minutes. The slides were left to cool in heated buffer for 20 minutes. Rinses and washes in PBS were carried out in between the following steps. Normal horse serum (50% in PBS) was used to non-specific binding of secondary antibody and blocked for 5 minutes. The FGF2 primary antibody was raised in a final dilution of (goat, 1 in 200 and 1 in 2000), control and IgG (a negative control) was applied to the slide covering the placental specimen with different dilutions and optimistised. The slide was incubated with primary antibody (rabbit, 1 in 1000) dilution for PLGF, and CD105, primary antibody (goat, 1 in 50 dilution) for HB-EGF, primary antibody (rabbit, 1 in 200 dilution) for MK and primary antibody (goat, 1 in 100 dilution) for PTN and control incubated overnight at 2-8°C. The next steps of this protocol are described below.
2.5.3. The VEGFR-1 (sflt-1)/ PLGF, VEGFR2 and FGFR-1 receptors.
The primary antibody was applied to the slide covering the placental specimen in the presence of citrate buffer with different dilutions and optimised. These slides were all prepared the same day for VEGFR-1 (sflt-1)/ PLGF receptor primary antibody (goat, 1 in 200 dilution), VEGFR-2 primary antibody (goat, 1 in 400 dilution) and FGFR-1 primary antibody (goat, 1 in 200 dilution) and control. After preparation of slides the sections were deparaffinised with 2 changes of xylene of 5 minutes each. In 100% alcohol for 3 minutes, 90% alcohol for 3 minutes, and 70% alcohol for 3 minutes. Then, the slide was blocked with 50% in PBS normal serum for 5 minutes. The slide was incubated with primary antibody rabbit for VEGFR-1 (sflt-1)/ PLGF (1 in 200 dilution), primary antibody VEGFR2 (1 in 400 dilution) and primary antibody goat FGF2 (1 in 200 dilution) and control for 2 hours. Then, secondary antibody was applied for 30 minutes for VEGFR-1 (sflt-1)/ PLGF and VEGFR-2 at room temperature. For FGFR-1 the secondary antibody was applied for 45 minutes at room temperature. The sections were incubated in preheated citrate buffer with 1M Tris to at pH 8.0 with HCL, and 0.5M EDTA for 10 minutes in a microwave and boiled in a microwave for 4 minutes. The slides were left to cool in heated buffer for 20 minutes. Rinses and washes in PBS were carried out in between the following steps. The next steps of this protocol are described below.
The diluted secondary biotinylated antibody (anti-rabbit Ig G (H+L) or anti-goat Ig G (H+L), 1 in 100 dilution) was applied for 30 minutes at room temperature. The section was incubated with a complex of avidin, labelled with horseradish peroxidase, and biotin (ABC) was applied for 20 minutes at room temperature. The slide was developed with 3, 3’-Diaminobenzidene (DAB) and hydrogen peroxide for 5 minutes followed by a counter staining in Mayer’s Haematoxylin solution for 10 minutes. Next the slide was differentiated in 1% acid alcohol for a few seconds. The slides were washed in running tap water for 5 minutes and followed the dehydration procedure. The slides were incubated in 70% alcohol for 2 minutes, 90% alcohol for 2 minutes, and 100% alcohol for 2 minutes, and in 2 changes of absolute alcohol for 3 minutes each. Finally, the slide was cleared in 2 changes of xylene, 3 minutes each. The slide was mounted in xylene -based mounting medium. The slide was then ready for examination under the Ceti microscope.