Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

First trimester human placental tissue of 7 to 12 weeks gestational age from 10 individuals was obtained from elective termination of uncomplicated pregnancies at Addenbrooke’s Hospital, Cambridge, with the permission of the local Ethical Committee. After collection, the tissue was dissected under culture media to identify the placental bed. The placental bed was difficult to identify macroscopically. Tissue close to area of yellow pigmentation, representing the Nitabuch’s Stria were randomly selected.

Tissue (< 1 mm in one dimension) was fixed by immersion in 4% formaldehyde (BDH Merck Ltd, Germany) in 0.1 M PIPES (Sigma Chemicals Ltd, Dorset, UK) buffer pH 7.6 for one hour at 4oC for light microscopy and immunocytochemistry or, in 4% glutaraldehyde (Taab Laboratories Ltd, Aldermaston, Berkshire, UK) in 0.1 M PIPES buffer pH 7.6 for one and a half hours at 4oC for ultrastructural studies. Tissue selected for light microscopy was either encapsulated in OCT (Tissue-Tek, Agar Scientific Ltd, Stanstead, Essex, UK) and frozen, or embedded in paraffin wax.

Cryostat sections (5 um in thickness) from each batch of formaldehyde-fixed tissue were stained with polyclonal rabbit anti-cytokeratin (ICN Biomedicals Ltd, Thame, Oxfordshire, UK) to confirm the presence of trophoblast. On this basis, the decidua was classified as decidua basalis if staining for cytokeratin was present or decidua parietalis if it was absent.

Light Microscopy
Following fixation, tissues were cryo-protected in 20% sucrose and incubated for one hour at 4oC in the fridge prior to sectioning. They were then frozen in isopentene cooled with liquid nitrogen. The frozen tissue was placed on a metal chuck containing a drop of OCT embedding compound and cooled at -26⁰C in a cryostat chamber. The tissue was then sectioned using Reichert Jung CM3000 at a thickness of 5 um and the sections thawed-mounted on glycerine coated glass slides. The sections were then immunostained for cytokeratin.

Immunohistochemistry
Paraffin sections of tissue were stained for cytokeratin and neutrophil elastase (NP57)(Dako Ltd, High Wycombe, UK) or leucocyte common antigen (Dako) or natural killer cell marker-CD56 (Dako) or macrophage marker-HAM-56 (Dako). They were dewaxed in xylene and rehydrated in series of ethanol concentrations. Endogenous peroxidase activity was blocked by treating the sections with 3% hydrogen peroxide in methanol for 20 minutes. The sections were then incubated in blocking buffer containing 2% normal rabbit serum in 0.01 M Tris-HCl buffered saline (TBS) at pH 7.6 to suppress non-specific binding. They were then incubated with polyclonal rabbit anti-cytokeratin antibody diluted 1:100 in blocking buffer in a humidified chamber for 30 minutes at room temperature. After rinsing extensively with TBS, the sections were incubated for a further 30 minutes with a biotinylated secondary goat anti-rabbit antibody (Vector Laboratories Ltd, Adermaston, Berkshire, UK) diluted 1:100 in the blocking solution. The sections were then rinsed thoroughly in TBS and subsequently incubated in streptavidin/biotin-horseradish peroxidase complex (Vectastain Elite ABC reagent) for 30 minutes. Antibody binding sites were visualised by incubation in 1 mg/ml 3’3 diaminobenzindine plus 0.03% (v/v) H2O2. This chromogen produced a brown reaction product.

After thorough rinsing in tris buffer, some sections were blocked with 2% normal rabbit serum for 15 minutes and incubated with mouse-monoclonal anti-human neutrophil elastase antibody or anti-human leucocyte common antigen or natural killer cell marker-CD56 or macrophage marker-HAM-56 diluted 1:100 in blocking buffer. After rinsing with TBS, sections were incubated with biotinylated rabbit anti-mouse secondary antibody diluted 1:100 in 2% normal rabbit serum for 30 minutes. The sections were then rinsed thoroughly in TBS buffer pH 7.6 and incubated in streptavidin/biotin-alkaline-phosphatase complex (Vector) for 30 minutes. New Fuschin Substrate System (Dako) was used for the visualisation of antibody binding sites to neutrophils and/or maternal leucocytes. The red colour of the final reaction product was clearly distinguishable from the brown reaction product of 3'3-diaminobenzidine used to label cytokeratin. The regions of necrosis in the test sections provided an internal positive controls for neutrophil elastase. The decidua for CD56, and sections of human tonsils for HAM-56.

Negative controls included omission of primary antibody with replacement of non-immune serum, followed by all of the subsequent steps. Non-specific labelling was negligible.

Ultrastructural Study
Tissues were fixed in 4% glutaraldehyde in 0.1 M PIPES buffer pH 7.6 for 1 hour at 4oC. They were washed in three changes of 0.1 M PIPES buffer pH 7.6, post-fixed in 1% osmium tetroxide (BDH) in 0.1 M PIPES at pH 7.6 containing 1.5% potassium ferricyanide (BDH) and 2 mmol/l calcium chloride for one hour at room temperature. They were then washed in 0.1 M PIPES buffer pH 7.6 for 2 minutes followed by 0.05M sodium maleate buffer, pH 5.2 for 10 minutes at 20^oC. They were then immersed in 2% uranyl acetate (BDH) in 0.05 M sodium hydrogen maleate buffer for one hour. After rinsing in distilled water they were dehydrated in an ascending series of ethanol solutions, treated with propylene oxide (Agar Scientific Ltd, Stanstead, Essex, UK), 2 changes of 15 minutes each, and transferred to a 50:50 mixture of propylene oxide and Spurr's resin (Taab Laboratories). They were agitated at room temperature overnight. This was followed by 8 hours in 25:75 propylene oxide and Spurr’s resin and four changes of 100% Spurr’s resin over 36 hours. They were transferred to latex moulds filled with resin and thermally cured at 60^oC for 48 hours. Sections 0.5 - 1 um were cut using a Reichart Ultracut S and stained with methylene-blue (BDH) for light microscopy. Areas of interest were identified and areas of necrosis selectively avoided. Ultra-thin sections of about 50-70 nm (showing silver to silver-grey interference colours) were cut and mounted on copper grids (Guilder, UK). They were double stained with uranyl acetate and lead citrate (BDH) for 30 seconds each and examined in a Philips CM100 at an accelerating voltage of 80 kV.

Freeze Substitution and Low Temperature Embedding
Tissues fixed in 4% formaldehyde were cryo-protected in 30% propylene glycol for one hour at 4^oC. They were then freeze substituted against methanol containing 0.1% uranyl acetate at -90^oC for 24 hours, -70C for 24 hours and -50^oC for 24 hours. The tissue was then impregnated with Lowicryl HM 20 over a period of 3 days and the resin was polymerised by ultraviolet irradiation at a temperature of -50^oC. Sections (50-70 nm) were cut and mounted on Formvar coated nickel grids.

Immunogold Labelling
Thin sections (50-70 nm in thickness) of placental bed, mounted on nickel grids were incubated overnight in primary antibody, rabbit anti-hPL (Dako) or mouse anti-cytokeratin (Dako) diluted in tris buffer containing 2% bovine serum albumin, 5% normal goat serum and 0.01% Tween 20 at pH 7.6. Postive control sections of placental villi were labelled for hPL and genital warts for cytokeratin. After extensive rinsing in tris buffer, the sections for hPL were floated on drops of biotinylated goat anti-rabbit antibody (Vecta) and for cytokeratin on rabbit anti-mouse antibody (Dako) diluted 1:100 for 1 hour at room temperature. After further rinsing, the sections were incubated for 1 hour at room temperature on drops of streptavidin conjugated to 1 nm gold particles (British Biocell Ltd, UK) diluted 1:100, and finally silver enhanced (IntenseEm, Amersham International, UK) for 4 minutes to visualise the gold particles. The sections were contrast stained in uranyl acetate and lead citrate for 30 seconds each and viewed in a Philips CM100 electron microscope at an accelerating voltage of 80 kV.

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