Supplementary Methods
Screening for the C9orf72 hexanucleotide repeat sequence by repeat primed PCR
A modified version of the repeat primed PCR method reported by Renton et al. (2011) was used
to identify cases. 100ng of genomic DNA was added to 5µl of FastStart PCR Master PCR
(Roche Diagnsotics Ltd, Burgess Hill, UK), 0.71ul of primer mix (40mM FAM labelled F-primer,
5mM R-primer, 40mM Anchor primer), 0.71ul of dimethyl sulphoxide (Sigma-Aldrich Company
Ltd, Dorset, UK), 1.79ul of 5x Q-solution (Qiagen Ltd, Crawley, UK), 0.36ul of 7-deaza-dGTP
(Roche Diagnsotics Ltd, Burgess Hill, UK), 0.36ul 25mM MgCl2 in a final reaction volume of
11ul. Samples were thermal cycled for 15min at 95˚C, followed by 7 sets of cycles of varying
lengths “Y” in which samples are denatured at 94˚C for 1min, annealed at variable annealing
temperature “X”for 1min and extended at 72 ˚C for 3mins. In set 1, X = 70˚C and Y = 2cycles; in
set 2, X decreases by 2˚C and Y increases by 1cycle such that by set 7, X = 58˚C and Y =
8cycles. Set 7 is followed by a final set of cycles ‘Set 8’ consisting of 10 cycles of 94˚C for 1min,
56˚C for 1min, 72˚C for 3mins. The reaction is completed by a final extension step of 72˚C for
10min. 2µl of the PCR products were added to 7.5µl of HiDye and 0.5ul of GS500LIZ (Applied
Biosystems, Life Technologies Corporation, California, USA) and analysed on an ABI 3730
capillary DNA analyser using a 60s injection time. Fragment data was analysed using peak
scanner software (Applied Biosystems, Life Technologies Corporation, California, USA).
Antibody
Isotype
Dilution
Antigen retrieval
Source
p62
Monoclonal
1:200
Menarini Diagnostics Access
BD (610833)
(3/P62 LCK Ligand)
TDP-43
FUS/TLS
OPTN
Polyclonal
polyclonal
polyclonal
unit/Revelation buffer(pH6.5)
1:200 to
Menarini Diagnostics Access
1:400
unit/Revelation buffer(pH6.5)
1:100
Menarini Diagnostics Access unit/Super
Novus via Stratech
buffer (pH8.0)
(NB100-2599)
Menarini Diagnostics Access unit/Super
Abcam (ab79110)
1:50
Proteintech (10782-2-AP)
buffer (pH8.0)
CD68
Monoclonal (KP1)
1:100
Menarini Diagnostics Access
Dako (M0814)
unit/Revelation buffer (pH6.5)
C9ORF72
Polyclonal
1:400
Menarini Diagnostics Access unit/Super
Santa Cruz Biotechnology
buffer (pH8.0)
(sc-138763
Supplementary Table 1: Antibody source and conditions for immunohistochemistry. All primary antibody incubations were for 30 minutes at
room temperature.