Immunostaining and Imaging

3.8.1 CryoSectioning

Human tissue samples were collected by the relevant tissue bank, snap frozen in liquid nitrogen and stored at -80 °C. CryoSectioning was performed by mounting the tissue block in optimum cutting temperature formulation (OCT) and allowing the sample to adjust to -20 °C in the cryostat chamber. Sections were cut 10 μm thick, collected on Superfrost glass slides and allowed to dry for 30 minutes at room temperature to avoid freezing artefacts.

3.8.2 H&E staining of human tissue Sections

Slides were washed for 2 minutes in dH2O and were placed in Haemalum solution for

1 minute. Slides were washed again for 2 minutes in dH2O and placed in Eosin solution

for 1 minute. Slides were washed for 2 minutes in dH2O before being dehydrated in

graded alcohol: 70% EtOH 30 seconds, 95% EtOH for 30 seconds, 100% EtOH for 2 minutes. Sections were covered in DPX solution and a glass coverslip was placed on top.

3.8.3 Immunostaining human tissue sections

Tissue sections on slides were fixed using 4% paraformaldehyde (PFA, Sigma Aldrich, Dorset, UK Aldrich) for 10 minutes. Slides were washed 3 times with PBS to completely remove the PFA. Sections were permeabilised by adding 0.5% Triton X-100/PBS for 10 minutes. After washing 3 times with PBS the Sections were blocked in buffer (5% FBS/0.1% Tween20/PBS) for 1 hour at room temperature. Primary antibodies were diluted in 2% FBS/PBS and were added to the Sections and left to incubate at 4 °C overnight.

Sections were washed twice for 5 minutes with cold PBS before the addition of the secondary fluorochrome-conjugated antibody diluted in 2% FBS/0.1% Tween20/PBS. Slides were incubated for 1 hour at room temperature in the dark. Slides were washed 3 x 5 minutes in PBS. Each Section was covered with VECTASHIELD Anti-fade Mounting Medium with DAPI (Vectorlabs).

3.8.4 Immunostaining cultured cells

Cells were grown on glass coverslips pre-coated with poly-L-lysine (Sigma Aldrich, Dorset, UK). Briefly, coverslips were washed in 70% EtOH before being placed into the well of a 6 well tissue culture plate and subjected to UV light for 20 minutes. 1 mL

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poly-L-lysine was added to each well to completely cover the surface area. The plate was incubated at room temperature for 30 minutes. The poly-L-lysine solution was removed and the wells were washed twice with sterile PBS. Plates were covered with parafilm and stored at 4 °C until required or were used immediately by washing once with DMEM before cells were plated.

When the cells were ready to image, growth media was removed and cells were fixed in 4% PFA at room temperature for 10 minutes. Cells were then washed twice with PBS for 2 minutes each time. Blocking buffer (PBS/5% normal goat serum/0.3% Triton X-100) was added to the cells for 1 hour at room temperature before the addition of primary antibody in dilution buffer (PBS/1% BSA/0.3% Triton X-100) overnight at 4 °C. Cells were washed three times with PBS before addition of the secondary fluorochrome- conjugated antibody for 1 hour in the dark. Cells were washed three times with PBS before the addition of VECTASHIELD Anti-fade Mounting Medium with DAPI

(Vectorlabs). All antibodies used are detailed in Table 3.5.

Antibody Manufacturer Catalog # Dilution Anti-PRDM9 Abcam, Cambridge, UK ab85654 1:500 Anti-FLAG M2 Sigma Aldrich F1804 1:500 Anti-OPA1 Abcam, Cambridge, UK ab42364 1:250 Anti-TFAM Source Bioscience, Manchester, UK LS-C143233 1:250 Anti-HSP60 Abcam, Cambridge, UK ab46798 1:400 Anti-TOMM20 Santa Cruz, Dallas, Texas, USA sc-17764 1:1000 Goat anti-mouse

Alexa Fluor® ₄₈₈

Fisher Scientific, Loughborough, UK A-11001 1:1000

Goat anti-rabbit Alexa Fluor® ₄₈₈

Fisher Scientific, Loughborough, UK A-11034 1:1000

Goat anti-mouse Alexa Fluor® ₅₉₄

Fisher Scientific, Loughborough, UK A-11032 1:1000

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Cells were imaged using the Nikon Confocal Microscope and or the AxioImager (Zeiss).

3.9 Cloning

Cloning was performed using the pGEM-T easy vector system (Promega, Southampton UK). Incubations carried out at 37 °C were performed in a temperature controlled microbiology incubator (Thermo Fisher Scientific, Loughborough, UK).

3.9.1 Product preparation

cDNA sequences of interest were provided already integrated into a vector DNA

backbone. In order to subclone insert sequences into another vector system, restriction enzymes (Table 3.6) were utilised to digest the insert from the pcDNA3.1(+) vector sequence. Reaction mixture was as follows: 50 ng plasmid DNA, 10 U each restriction enzyme, 2 μL CutSmart buffer (New England Biolabs inc.), 3 μL10X BSA made up to a final volume of 50 μL with dH2O. Reactions were incubated at 37 °C for 1 hour. The total

volume of digested sample was run on a size selection gel as previously described. The product of interest was gel extracted using the QIAquick gel extraction kit following manufacturers instructions (Qiagen, Manchester, UK, Manchester UK).

Restriction enzyme Manufacturer 5’-3’ Cut site ApaI New England Biolabs Inc. GGGCCC BamHI New England Biolabs Inc. GGATCC EcoRV New England Biolabs Inc. GATATC HindIII New England Biolabs Inc. AAGCTT PmeI New England Biolabs Inc. GTTTAAAC XhoI New England Biolabs Inc. CTCGAG

Table 3.6 List of restriction enzymes used in this study.

3.9.2 Ligation

Next, a ligation reaction was set up using the ligase provided in the pGEM-T easy kit (Promega, Southampton UK). Reaction mixture was as follows: 2X Rapid ligation buffer,

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50 ng pcDNA5 vector, 3U T4 DNA ligase, dH2O and the volume of insert DNA from the

restriction enzyme digest as calculated by the following equation:

𝑖𝑛𝑠𝑒𝑟𝑡: 𝑣𝑒𝑐𝑡𝑜𝑟 𝑚𝑜𝑙𝑎𝑟 𝑟𝑎𝑡𝑖𝑜 = 𝑛𝑔 𝑣𝑒𝑐𝑡𝑜𝑟 𝑥 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑖𝑛𝑠𝑒𝑟𝑡 (𝐾𝑏) 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑣𝑒𝑐𝑡𝑜𝑟 (𝐾𝑏)

To maximise the number of positive transformants from the reaction, ligation was performed at 16 °C for 12 hours.

3.9.3 Transformation

Ligation transformation was performed using JM109 high efficiency competent cells (Promega, Southampton UK), 2X Yeast Tryptone broth (2YT) and 2YT agar plates were used as growth medium. 2YT agar was autoclaved and allowed to cool to approximately 55 °C before being supplemented with 100 mg/mL ampicillin, 75 μg/mL X-Gal and 0.5 mM IPTG. Agar was poured into 90 mm plates under aseptic conditions and left to cool and solidify at room temperature. Plates were either dried at 37 °C or stored at 4 °C for up to 1 month. 5 μL of ligation product was added to pre-aliquoted (25 μL)

competent cells on ice, gently agitating the tube to ensure the ligation product and cells were mixed. Competent cells used in this study are detailed in Table 3.7. The mixture was incubated on ice for 20 minutes before being subjected to heat shock for 45 seconds at 42 °C using a dry heat bath with 200 μL water added to the well. The tube was then returned to ice for 2 minutes before 470 μL autoclaved 2YT broth was added to the transformants and incubated at 37 °C for 1 hour at 150 rpm. To plate the cells, 200 μL of the cell suspension was spread onto an agar plate under aseptic conditions using a glass spreader dipped in ethanol and flamed. Plates were incubated for 16 hours at 37 °C. White colonies were picked for colony PCR and sequencing. Growth of white colonies indicates a successful ligation and transformation. Blue colonies indicate a successful transformation but inefficient ligation due to the activation of β-galactosidase. To perform the PCR 10 μL dH2O was added to each tube in a sterile 8 strip PCR tube.

Autoclaved pipette tips were used to pick single colonies from the growth plate and were dipped in the PCR tube before being streaked onto a new growth plate and placed in 5 mL fresh 2YT broth, supplemented with 100 mg/mL ampicillin. The plate was

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incubated at 37 °C to expand the colonies and the 5 mL inoculated 2YT broth was incubated at 37 °C at 150 rpm for 16 hours. Due to the size of the insert DNA sequences used in this study, long range PCR was performed as follows; to the 10 μL dH2O colony

pick, 10% betaine, 10 %10X LA PCR Buffer II (Mg2+_{), 200 μM dNTP, 0.2 μM forward}

primer, 0.2 μM reverse primer and 1.25 U TaKaRa LA Taq was added to a final reaction volume of 25 μL. Thermocycling conditions were performed in 30 cycles of; 94 °C for 1 minute, denaturing at 98 °C for 10 seconds, primer annealing at 62 °C for 15 minutes followed by final extension at 72 °C for 10 minutes. Sequencing was performed as

previously described. Once the desired product was confirmed by running PCR products on size separation gels and sequencing, 1 mL of the 5 mL inoculated broth was used to inoculate 100 mL 2YT broth. This broth was incubated at 37 °C for 16 hours at 150 rpm. The remaining 4 mL of original inoculated broth was used to create glycerol stocks of the colony, 150 μL glycerol was added to 850 μL broth in a 2 mL cryovial and stored at -80 °C.

Competent Cell Type Manufacturer Application JM109 Promega Plasmids <10Kb

One Shot® _{Stbl3™ Invitrogen Large unstable plasmids} One Shot® MAX Efficiency® DH5α™ Invitrogen Large plasmids

Table 3.7 Details of all competent cell types used throughout the study.

3.9.4 Plasmid purification

Growth medium inoculated with colonies carrying the correct plasmid and insert was centrifuged for 15 minutes at 6000 x rcf at 4 °C. Pellets were then processed using the Qiagen, Plasmid Maxi Kit (Qiagen, Manchester, UK) according to the manufacturers instructions. The final pellet was air dried for 10 minutes to allow any remaining ethanol to evaporate and the DNA was resuspended in 500 μL dH2O. DNA concentration was

measured using the Nanodrop2000 UV-vis Spectrophotometer (Thermo Fisher Scientific, Loughborough, UK).

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