IMMUNOHISTOCHEMISTRY

2.6.1 Fixation of tissue for immunohistochemistry

Small pieces of myometrium approx 0.5mm x 0.5mm x 0.5mm were dissected and placed into neutral buffered formalin containing 10 % formalin. The fixed samples were left for 24 hours in the physiology fridge before transferring back to Liverpool Women’s Hospital. The fixed samples were processed and embedded in paraffin wax by Kelly Harper at Liverpool Women’s Hospital for human sections, and animal sections in the pathology department in the University of Liverpool Veterinary School and 4μm sections were cut, then mounted onto glass slides. Sections were cut from 6 pregnant and non-pregnant women as well as different stage gestation rats. For control tissues and antibody optimisation animal tissue was also used. Aorta and Kidney were dissected from 22 day pregnant Wistar rats, also non- pregnant, 10, 14, 18, 22 day as well as labouring rat uterus were dissected. They were then placed in Formalin solution, neutral buffered, 10%. Embedding and sectioning was carried out in the pathology department in the University of Liverpool Veterinary School.

2.6.2 Staining for CBS and CSE

CBS enzyme – CBS monoclonal antibody, Abnova, clone 3E1 : 1:50 CSE enzyme- CTH monoclonal antibody, Abnova, clone 4E1-1B7 : 1:150

Both CBS and CSE antibodies have been used multiple times in recent literature (Rashid 2012, Kasparek 2011, Fu 2012).

Beta Actin - beta Actin antibody, Abcam : 1:500

Sections from human myometrium were labelled appropriately before combining in a metal rack. The rack was placed into a glass bath containing 100% Xylene for 30minutes to remove the paraffin wax from the section. The sections were then dipped in a series of ethanol baths from 100%. 95%, 85%, 70% and 50% in order to rehydrate the sample before being placed into a bath of distilled water for 5 minutes. The slides were then placed into a bath of boiling 14mM Sodium Citrate buffer pH6.0 in a microwave for 20mins to allow optimum antigen retrieval. The slides were allowed to cool for 20minutes on ice before washing with distilled water. Each slide was removed of excess water before circling the tissue section with

93 ImmEdge Hydrophobic barrier pen (Vector Laboratories) which allows reagents to remain localized. To block endogenous peroxidise activity 3% hydrogen peroxide (30% Hydrogen peroxide in tris buffered saline (TBS) solution) was dropped onto each tissue section and incubated for 30 minutes in a humid chamber. Slides were then rinsed with TBS-T (0.05% Tween20) and incubated for 1hour with non specific block 5% Bovine Albumin Serum (BSA) dissolved in 0.05% TBS-T. Slides were then rinsed again in TBS-T before overnight incubation with the appropriate antibody in blocking solution at 4°C. As a negative control one section from both non-pregnant hysterectomy and term pregnant patients, as well as a multiple stage rat myometrium slide, were incubated overnight in blocking solution alone. As a positive control an animal section that is known to express the protein of interest was used and for an antibody control beta actin was used.

The following day the primary antibody was removed by 3 X 15 minute washes with TBS-T

0.05% before incubating with secondary antibody at room temperature for 1hour (impress

Universal Antibody anti-mouse Ig /anti-rabbit Ig, peroxidise polymer detection kit, Vector Labs). The secondary antibody was then removed by 3 X 15minute washes with TBS-T

0.05%. As a HRP labelled secondary was used 3,3'-Diaminobenzidine (DAB) was used for developing (3,3′-Diaminobenzidine (DAB) Enhanced Liquid Substrate System tetrahydrochloride, Sigma). DAB was dropped onto the positive control slide first and placed under a microscope until a brown colour developed. The reaction was stopped by placing slide into distilled water. All slides from pregnant and non- pregnant human were developed for the same time to reduce any bias in the experiments; the same was done for the different stage gestation animal tissue. Once all slides were developed the slides were counterstained with Haematoxylin by incubating with the stain for 1 minute followed by washing with tap water for 5 minutes. The slides were then dehydrated by dipping in a series of ethanol baths from 50%, 70%, 85%, 95% and 100% before being placed into xylene. This removed any excess paraffin pen that was encircling the section. Cover slips were then mounted using DPX mountant and left to dry overnight before image capture.

94 2.6.3 Image Capture of Myometrial Biopsy Sections – DAB Stained.

The setup used was provided by Liverpool Women’s Hospital university department and was composed of the following -

1. Nikon Eclipse 50i Microscope, Nikon Corporation, Tokyo 100-8331, Japan

2. Nikon DS-Fi1 digital camera Head 5M pixel, Nikon Corporation, Tokyo 100-8331, Japan

3. Nikon Digital control unit DS-U2 USB, Nikon Corporation, Tokyo 100-8331, Japan 4. Nikon C-Mount TV adaptor, 0.63x, Nikon Corporation, Tokyo 100-8331, Japan 5. NIS-Elements-F software, developed for Nikon Instruments

6. Personal computer (minimum specification 1GB RAM, 2.8GHz processor)

After launching the NIS- Elements-F software a slide was placed on the microscope stage and moved to an area that contained a section of myometrial biopsy. The settings were adjusted to normal mode, 640x480 resolution, 1280x960 quality capture, and high colour contrast and sharpness. The microscope was calibrated using a scale slide for each objective

– 4x, 10x and 40x. The whole tissue section was scanned on 4x objective and 10 x objective to examine which areas would be most representative of the entire tissue for photographing. The negative control was examined first to make sure there was no staining

– if any staining was present then the experiment would have to be repeated. All slides were blinded to the observer to reduce any bias. After locating the areas with a high amount of staining the 40x objective was used to photograph 10 different areas of the section. This was repeated for all sections in each immunohistochemistry.

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