Immunochemistry and Microscopy

Immunochemistry is a method typically employed to detect a particular protein of interest in cells (immunocytochemistry, ICC) and tissue (immunohistochemistry, IHC) via the use of fluorescent-labelled antibodies which bind to their target antigens. This technique enables researchers to visualise the localisation and morphology (i.e. the size and shape of the cell) of individual cell components which are maintained during immunochemistry procedures (Coons

et al., 1942).

Samples are first ‘fixed’ using crosslinking reagents (e.g. formaldehyde/formalin) which form bridges or organic solvents (e.g. methanol and acetone) to dehydrate cells and remove lipids in order to retain cytoskeletal morphology. Specific reagents such as Triton X-100 are then used for permeabilising the cell membrane, enabling antibodies to penetrate into the sample and bind to the antigen of interest. Serum, specifically that derived from the same species in which the secondary antibody was raised is used for ‘blocking’, preventing any unwanted binding of the primary antibody to non-specific sites within the sample which may lead to background staining. Permeabilising and blocking are usually undertaken together for time efficiency.

There are two major classes of antibodies used within immunochemistry, namely polyclonal and monoclonal antibodies. Polyclonal antibodies are raised in-vivo, whereby animals are injected with the antigen of interest which allows B lymphocytes to produce quantities of the antibody which are later purified from the serum. Similarly, monoclonal antibodies are generated via immunising the host with the antigen. However, hybridomas derived from the fusion of lymphocytes of the animal spleen and myeloma cells are expanded in culture, forming a single clone (Köhler & Milstein, 1976). Monoclonal antibodies have high specificity as they are epitope specific and may therefore be considered preferable for therapeutic drug

development. However, polyclonal antibodies have a higher antibody affinity as they recognise multiple epitopes on a given antigen, suggesting a greater robust protein detection. The use of either antibody may therefore be determined based on the application of interest.

Once the samples are permeabilised and ‘blocked’, a primary antibody is added which binds to the protein/antigen of interest. Following primary incubation, the antibody is removed, and the sample is washed several times. A secondary antibody conjugated to a fluorophore (fluorescent dye), which is raised against the host species in which the primary antibody was raised, is then added to the sample to enable detection of the antigen using fluorescence microscopy by absorbing and emitting light at specific wavelengths.

2.7.2! Immunostaining Human Skeletal Muscle Derived Cells

Cells within 6-well plates were washed 3 × TBS (1×; Sigma-Aldrich, UK) and fixed and dehydrated in ice-cold methanol:acetone:TBS (25:25:50) for 15 mins then a further 15 mins in methanol:acetone (50:50) only. Following 3 × further washes, 6-well plates were wrapped in parafilm and stored at 4˚C until required for immunostaining.

Following fixation, cells were permeabilised in 0.2% Triton X-100 (Sigma-Aldrich, UK) and blocked in 5% goat serum (Sigma-Aldrich, UK) in TBS for 90 mins. Cells were then washed 3 × in TBS and incubated overnight (4˚C) in 300 µl of anti-desmin (ab15200, Abcam, UK) primary antibody made up in TBS, 2% goat serum and 0.2% Triton X-100 at concentrations of 1:50. After overnight incubation, the primary antibody was removed and cells were washed 3 × in TBS. Cells were then incubated at RT for 3 hrs in 300 µl secondary antibody solution containing anti-rabbit TRITC (T6778, Sigma-Aldrich, UK) at a concentration of 1:75 in 1× TBS, 2% goat serum and 0.2% Triton X-100 to counterstain myoblasts. After a further 3 × TBS washes, 300 µl of DAPI (D1306, Thermo Fisher Scientific, Denmark) was added to the cells

at a concentration of 300 nM for 30 mins to counterstain myonuclei. Once stained, 2 ml of TBS was added to each well and culture plates were sealed with parafilm and covered with foil and were stored at 4˚C until required for fluorescence imaging.

2.7.3! Immunostaining Bioengineered Skeletal Muscle

Bioengineered SkM constructs were fixed using methanol and acetone as described in section 2.7.2. The fixation method used will be described within the methods section of each experimental chapter throughout this thesis. Following fixation, pins were removed, and constructs were transferred to 2 ml Eppendorf tubes using 2 × sets of angled tweezers (see Figure 2.13). Gels were then permeabilised (0.2% Triton X-100) and blocked (5% goat serum) in TBS (1×) for 90 min and incubated overnight (4˚C) in 250 µl of Phalloidin-FITC antibody (P5282, Sigma-Aldrich, UK) at a concentration of 50 µg/ml. After overnight incubation, the antibody was aspirated and gels were washed 3 × in TBS before adding 250 µl of DAPI (300 nM) for 90 mins to counterstain myonuclei. When immunostaining for desmin, antibody concentrations and incubation durations were the same as that described in section 2.7.2 for HMDCs cultured in 6-well plates. Once stained, muscle constructs were transferred to non- sylgard coated culture dishes containing 2 ml of TBS and were wrapped in parafilm and foil and stored at 4˚C until required for fluorescence imaging.

Fixed SkM constructs (n = 4) were transferred to 2 ml Eppendorf’s held in place using blue tac. Windows were carved longitudinally to enable easy access of gels. Green circles highlight muscle constructs.

2.7.4! Microscopy and Image Analysis

Immunostained cells and SkM constructs were visualised using either an inverted fluorescence (Nikon, Eclipse Ti-S, Japan) or confocal (Olympus IX83, Japan) microscope and were imaged using corresponding software (Nikon, NIS Elements and FV10-ASW 4.2 for fluorescence and confocal microscopy, respectively). Microscopy equipment/procedures is detailed within the methods section of each experimental chapter. Myoblasts and myotubes were visualised using Figure 2.13. Immunostaining fibrin bioengineered SkM

FITC (Phalloidin-FITC, Excitation: 495 nm, Emission: 513 nm) and TRITC (Desmin, Excitation: 557 nm, Emission: 576 nm) filters and myonuclei was visualised using a DAPI filter cube (Excitation: 358 nm, Emission: 461 nm). All immunostained samples were imported to Fiji/ImageJ (version 2.0.0) software for subsequent analysis. Primary heterogenous cell populations were characterised via counting the total number of myoblasts (red) overlapping nuclei (blue) and dividing this by the total number of nuclei (see Figure 2.14).

Example of immuno-stained (A-C) and bright-field (D) microscopic images of HMDCs (Nikon, Eclipse Ti-S, 10× magnification scale bar = 100 µm) stained with (A) primary antibody only (anti-desmin), (B) secondary antibody (anti-rabbit TRITC) or (C) desmin (red) and Figure 2.14. Immunostaining of HMDCs for determining myogenic population

A

B

myonuclei (blue). Myogenicity (%) was calculated via dividing the sum of all desmin positive cells by the total number of blue/nuclei cells present.

2.8!Lysing and Homogenising Bioengineered Skeletal Muscle Tissue for RNA and