Immunocytochemistry

2.9.1.Immunocytochemical staining

2.9.1.1. Staining procedure

Media was removed from the cells and replaced with ice-cold acetone (dried with Molecular Sieve type 4A beads (ProSciTech, cat# C830)) for 5 minutes to fix cells. Following fixation, cells were washed with tris-HCl (0.5M, made up as described in section 2.12.1). Heat retrieval of the antigens was performed in High pH FLEX Target Retrieval Solution (pH9; Dako, cat# K8004) in a Dako PT-Link at 97°C for 15 minutes.

Following heat retrieval, slides were briefly rinsed with deionised water and placed in the Dako Autostainer Plus, running the following program:

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

90 Table 2.9-1: Dako autostainer program for immunocytochemistry.

Step name Reagent

(600μl) Time (min) Rinse Tris-HCl -- Permeablisation 3% H2O2 20 Rinse Tris-HCl -- Rinse Tris-HCl -- Primary antibody See Table 2.12-2 90 Rinse Tris-HCl -- Rinse Tris-HCl -- Mouse HRP Envision Mouse HRP 30 Rinse Tris-HCl -- Rinse Tris-HCl -- DAB+ DAB+ 10 Rinse Tris-HCl -- Rinse Distilled water --

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

91 For negative control slides, the primary antibody was replaced with an isotype control

consisting of species appropriate IgG. Slides were rinsed with water and placed in Meyer’s haematoxylin for 5 minutes. Excess haematoxylin was rinsed off under running tap water until the water ran clear rather than blue or purple. Haematoxylin staining was fixed by placing the slides into ammoniated water for a minimum of 7 minutes.

Following nuclear staining, slides were dehydrated by moving them through a series of dehydrating solutions. In order, the solutions were: 95% ethanol, 100% ethanol (two different batches), 100% xylene (two different batches). The slides were exposed to the initial three solutions (ethanol-containing) for five minutes each, and were exposed to the final two solutions (xylene) for two minutes per solution. The slides were manually mounted using Dako hard-set mounting media (Dako, cat# CS703), with unmounted slides left in the final xylene bath until mounting. Mounted slides were air-dried overnight at room temperature.

2.9.1.2. Imaging procedure

Slides were imaged using either an Olympus Virtual Slide Imager VS120 or an Axio Lab.A1 (Zeiss) microscope with attached AxioCam ICc5 (Zeiss). Images taken using the VS120 slide scanner utilised the bundled imaging software, while the AxioCam utilised Zen (Blue

Edition, v 2.3).

The Olympus scanner was set to take an image of size 6mm x 6mm from each well at 20x objective magnification. The Axio Lab.A1 was used at 20x objective magnification to image 16 non-overlapping fields of view.

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

92

2.9.2.Converting image files to usable formats and size

The Olympus Virtual Slide Imager VS120 created all image files in a proprietary filetype (.vsi), which was not readable by most imaging programs. Although FIJI Is Just ImageJ (FIJI) could open these files using the Bioformats plugin, due to the size of the images (>1GB) it was nearly impossible to analyse the images using available equipment.

To aid in analysis, a macro was written to firstly open every image (around 700-750 images) and convert it into the .tiff file format, allowing the images to be opened easily by FIJI without the use of the Bioformats plugin (see Appendix 1: FIJI and ImageJ Macros (Raw Code) for macro codes). A second macro was then written to take each .tiff file, convert it into RGB colour (to allow analysis of staining, discussed in the next section) and then select 16 non-overlapping fields of view and save each field of view as a separate image. The fields of view were selected at fixed locations spread across the whole image and were positioned identically in every image, to prevent bias when selecting areas to analyse. This produced more than 11,000 images.

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

93

2.9.3.Image analysis

Image analysis of immunocytochemical stainingstaining was performed using a combination of ImageJ and FIJI. From each well (16 images total), five images were randomly selected by random number generator (using the website https://www.random.org/) and the nuclei in these images were manually counted using the point selection tool in ImageJ or FIJI.

Initially (for 9 slides – a total of 360 images) the staining in each cell was measured utilising the ImageJ plugin IHCToolbox [210]. However the scale of the analysis required automation. Dr. Jo-Maree Courtney wrote a macro for FIJI which randomly selected five images from each well and opened them sequentially without user input. The nuclei could then be counted manually for each image, following which the macro would apply colour deconvolution to the image. Colour deconvolution is an in-built FIJI function which can separate DAB and haematoxylin staining into individual channels, allowing the user to utilise the ‘threshold’ function of FIJI to select and measure the area stained with DAB without selecting

haematoxylin-stained areas.

This macro was used to analyse the remaining images, although it required user-coded changes to accurately select the area stained and to ensure the proper scale was set for each image (see Appendix 1: FIJI and ImageJ Macros (Raw Code) for macro code). Data were expressed as area stained/cell, a measurement which was obtained by dividing the total area stained in an image by the number of nuclei present. Analysis of the data consisted of a two- way ANOVA applied to the three pHBEC groups treated with drugs, and all other data were analysed with one-way ANOVAs.

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

94