Supplement Table 1. Characteristics of the brain samples of the frontal cortex used for core samples to be assessed in the tissue microarray sections in Trial 1 (TMA-A: A1-A15), Trial 2 (TMA-B: B1-B15) and Trial 3 (TMA-B: C1-C15)
Coresin
TMA-A
(trial 1) / Cores
in
TMA-B
(trial 2) / Cores
in
TMA-B
(trial 3) / Gender / Age at death
(years) / Post-mortem
delay
(hours) / Fixative / Fixation
time
(days) / Embedding medium/
highest temperature / Storage
time
(months)
A1, A2 / - / - / F / 50 / 24 / 10% neutral phosphate-buffered formalin / 1,710 / Histosec Merck / 62°C / 0.25
A3, A4 / - / C5 / M / 75 / 6.5 / 4% formaldehyde / 256 / Paraffin / 60°C / 8.5
A5 / B6, B7 / C6, C7 / n.a. / 56 / 14 / 4% buffered formalin / 90 / Paraffin / 57°C / 4
A6, A7 / B10 / - / F / 69 / 24 / 10% buffered formalin / 210 / Paraffin wax / 62°C / 2.5
A8, A9 / B1 / C8, C9 / M / 73 / 13 / 4% formalin / 446 / Bayers paraffin / 57°C / 41
A10 / - / - / F / 87 / 24 / 10% Fischar unbuffered formalin / 35 / Histosec Merck / 63°C / 90
A11 / B11, B12 / C1 / F / 68 / 3 / 10% buffered formalin / 88 / White paraffin / 56°C / 129
A12, A13 / B13 / C2 / M / 73 / 12 / 10% buffered formalin / 37 / White paraffin / 56°C / 61
A14 / - / - / M / 57 / 24 / 10% buffered formalin / 69 / White paraffin / 56°C / 45
A15 / - / C3 / F / 46 / 24 / 4% formaldehyde / n.a. / n.a. / n.a.
- / B2, B3 / C10, C11 / F / 73 / 23 / 4% buffered formalin / 26 / Kendall paraplast plus / 60°C / 5
- / B4, B5 / - / M / 60 / 6.5 / 4% buffered formaldehyde / 41 / Histowax / 62°C / 140
- / B8, B9 / - / n.a. / 56 / 14 / 4% buffered formalin / 90 / Paraffin / 57°C / 4
- / B14, B15 / C13, C14 / F / 58 / 48 / 10% buffered formalin / 42 / White paraffin / 56°C / 35
- / - / C4 / F / 72 / 36 / 4% formaldehyde / n.a. / n.a. / n.a.
- / - / C12 / F / 70 / 38 / 10% buffered formalin / 56 / White paraffin / 56°C / 47
- / - / C15 / M / 67 / 28 / 4% formaldehyde / n.a. / n.a. / n.a.
- / - / - / M / 65 / 77 / 10% buffered formalin / 55 / White paraffin / 56°C / 43
F, female; M, male; n.a., not available; -, not selected
Supplement Table 2. Immunohistochemistry: Antibodies, antigen retrieval methods and detection systems in Trial 1
time / Detection reagent, (Code), Company1 / Chromogen / Evaluation
(Quality)
UBIQUITIN
Monoclonal
Mouse anti-Ubiquitin,
clone Ubi-1, (MAB1510),
Chemicon, Temecula, CA / M / 20 min in CB, pH 6.0 / 1:6,000 / +4°C, o/n / EnVision, (K4001), Dako / DAB / 12 (A)
M / AC/120°C 10 min in aqua / 1:100 / RT, 30 min / EnVision, (K5007), Dako / DAB / 19 (P)
M / AC/120°C 10 min in aqua / 1:50,000 / +4°C, o/n / PowerVision+ PolyHRP IHC Kit, (DPVB+110DAB), ImmunoVision Technologies / Romulin
AEC / 3 (G)
M / 4% FA, 15 min / 1:256,000 / +4°C, o/n / PowerVision, (DPVB-999HRP), ImmunoLogic / DAB / 17 (P)
Polyclonal
Rabbit anti-Ubiquitin, (Z0458),
DakoCytomation, Glostrup, Denmark / M / None / 1:200 / RT, 30 min / EnVision, (K5007), Dako / DAB / 18 (P)
M / None / 1:200 / +4°C, o/n / Vectastain Elite ABC, (PK6100), Vector / DAB / 26 (A)
M / None / 1:1,000 / +4°C, o/n / goat anti rabbit-AP coupled 1:100, (D0487), Dako / Neufuchsine / 31 (A)
M / Boiling 10 min in CB, pH 6.0 / 1:200 / RT, 25 min / EnVision, (K5007), Dako / DAB / 7,8,9 (A)
M / AC/120°C 10 min in CB, pH 6.0 / 1:200 / +4°C, o/n / Super sensitive IHC/HRP detection kit, (LP 000-UL), BioGenex / Romulin
AEC / 29 (A)
M / AC/120°C 10 min in aqua / 1:300 / RT, o/n / EnVision, (K4003), Dako / DAB / 11 (A)
M / AC/120°C 10 min in aqua / 1:500 / RT, o/n / EnVision, (K4003), Dako / DAB / 10 (A)
M / MW 3x5 min in CB, pH 6.0 / 1:1,000 / RT, 45 min / Vectastain Elite Kit, (PK-6101), Vector / DAB / 5 (P)
A / None / 1:100 / RT, 30 min / ABC, Zytochem Plus HRP-kit, (HRP500), Zytomed Systems / DAB / 25 (P)
A / CCl (Equivalent EDTA) / 1:500 / +37°C, 30 min / iVIEW™ DAB Detection, (760-091), Ventana Medical Systems / DAB / 27 (P), 28 (A)
A / 100% FA, 5 min / 1:500 / +37°C, 32 min / iVIEW™ DAB Detection, (760-091), Ventana Medical Systems / DAB / 30 (P)
p62
Monoclonal
Mouse anti-p62 lck ligand,
clone 3, (610833),
BD Biosciences Pharmingen,
San Diego, CA / M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / Histostain Plus Kit (85-9043), Zymed / Romulin
AEC / 32 (G)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +5°C, o/n / EnVision, (K5007), Dako / DAB / 20 (G)
M / MW 10 min in CB, pH 6.0 / 1:1,000 / +5°C, o/n / ABC Complex/HRP, (K0355), Dako / DAB / 4 (A)
M / MW in a Pressure cooker 15 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / ABC Complex/HRP, (K0355), Dako / Romulin
AEC / 21 (G)
A / Ventana CCl standard program with 12 min boiling up to 100°C / 1:100 / +37°C, 32 min / iVIEW™ DAB Detection, (760-091), Ventana Medical Systems / DAB / 1 (G)
A / MW 1 h in CB, pH 6.0 / 1:100 / RT, 1h / Dako REAL™, (K5001), Dako / DAB / 14 (G), 15 (G), 16 (G)
Polyclonal
Guinea Pig anti-p62 Protein (N terminus), (GP62-N),
Progen, Heidelberg, Germany / M / None / 1:100 / +4°C, o/n / Vectastain Elite ABC, (PK-6100), Vector / DAB / 6 (A)
TDP43/TARDBP
Monoclonal
Mouse anti-TARDBP,
clone 2E2-D3, (H00023435-M01),
Abnova, Taipei, Taiwan / M / AC/120°C 10 min in CB, pH 6.0 / 1:800 / +4°C, o/n / StreptABC complex/HRP, (K0377), Dako / DAB / 34 (P)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / Histostain Plus Kit (85-9043), Zymed / Romulin
AEC / 33 (P)
Polyclonal
Rabbit anti-Human TARDBP, (10782-1-AP),
ProteinTech Group, Chicago, IL / M / 5x3 min in CB, pH 6.0 / 1:300 / RT, 90 min / goat anti-rabbit-AP coupled 1:100, (D0487), Dako / Neufuchsine / 24 (P)
M / MW 3x5 min in CB, pH 6.0 / 1:2,000 / +4°C, o/n / Biotinylated swine anti-rabbit 1:500, (E0353), Dako & Vectastain Elite ABC (PK-6102), Vector / DAB / 2 (P)
A / MW 3x5 min (450w), in CB, pH 6.0 / 1:2,000 / +37°C, 32 min / iVIEW™ DAB Detection, (760-091), Ventana Medical Systems / DAB / 13 (P)
AC, autoclave; MW, microwave; CB, citrate buffer; FA, formic acid; o/n, overnight; RT, room temperature; A, acceptable; G, good; P, poor quality of staining
1 BioGenex, San Ramon, CA; Dako (DakoCytomation), Glostrup, Denmark; ImmunoLogic, Duiven, the Netherlands; ImmunoVision Technologies, Norwell, MA; Vector, Burlingame, CA; Ventana Medical Systems, Tucson, AZ; Zymed, South San Francisco, CA; Zytomed Systems, Berlin, Germany
Supplement Table 5. Immunohistochemistry: Selected antibodies and antigen retrieval methods as recommended in Trial 2. Slides used for the inter-rater
assessments are stated in bold.
Antibody, (Code), Company / Mode of staining / Pre-treatment / Dilution / Incubationtime / Detection reagent, (Code), Company1 / Chromogen / Evaluation
(Quality)
p62
Monoclonal
Mouse anti-p62 lck ligand,
clone 3, (610833),
BD Biosciences Pharmingen,
San Diego, CA / M / Steamer 30 min, in CB, pH 6.0 / 1:500 / RT, 90 min / goat anti mouse AP 1:500, (D0486), Dako / Neufuchsin / 24 (P)
M / AC/121°C 10 min in deionised water / 1:1,000 / +4°C, o/n / Vectastain ABC, (PK6200), Vector / DAB / 15 (A)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +5°C, o/n / Histostain Plus Kit (85-9043), Zymed / DAB / 28 (P)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / Super sensitive IHC/HRP detection kit, (LP 000-UL), BioGenex / Romulin
AEC / 29 (P)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +5°C, o/n / Vectastain Elite Standard ABC (PK-6100), Vector / DAB / 5 (A)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +5°C, o/n / Vectastain Elite ABC, (PK-6102), Vector / DAB / 1 (A)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / ABC Complex/HRP, (K0355), Dako / Romulin
AEC / 21 (G)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / Dako REALTM, (K5001), Dako / DAB / 39 (P)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / RT, o/n / EnVision, (K4003), Dako / DAB / 10 (G),11 (G)
M / AC/120°C 15 min, in CB, pH 6.0 / 1:1,000 / +20°C, 24 h / EnVision, (K4001), Dako / DAB / 25 (G)
M / MW 2x10 min (450w) in CB, pH 6.0 / 1:1,000 / +5°C, o/n / Dako REALTM, (K5001), Dako / DAB / 13 (G)
Polyclonal
Guinea Pig anti-p62 Protein
(N terminus), (GP62-N),
Progen, Heidelberg, Germany / M / None / 1:100 / +4°C, o/n / Vectastain Elite ABC, (PK-6100), Vector / DAB / 6 (G)
M / Boiling in steamer 1 h in CB, pH 6.0 / 1:4,000 / +5°C, o/n / EnVision, (K5007), Dako / DAB / 8,9,37 (A)
TDP43/TARDBP
Monoclonal
Mouse anti-TARDBP,
clone 2E2-D3,
(H00023435-M01),
Abnova, Taipei, Taiwan / M / AC/120°C 10 min in CB, pH 6.0 / 1:300 / +4°C, o/n / Streptavidin peroxidase, (K0690), Dako / DAB / 12 (A)
M / Boiling in steamer 1 h in CB, pH 6.0 & 100% FA 1 min / 1:2,000 / RT, 25 min / EnVision, (K5007), Dako / DAB / 22,35,38 (A)
Polyclonal
Rabbit anti-Human TARDBP, (10782-1-AP),
ProteinTech Group, Chicago, IL / M / AC/121°C 10 min in deionised water / 1:1,000 / +4°C, o/n / Dako REALTM, (K5001), Dako / DAB / 16 (G),14 (G)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / Dako REALTM, (K5001), Dako / DAB / 40 (A)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +5°C, o/n / EnVision, (K5007), Dako / DAB / 18,19,20 (A)
M / AC/120°C 10 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / Super sensitive IHC/HRP detection kit, (LP 000-UL), BioGenex / Romulin
AEC / 36 (A)
M / MW 3x5 min in CB, pH 6.0 / 1:2,000 / +4°C, o/n / Biotinylated swine anti-rabbit 1:500, (E0353), Dako & Vectastain Elite ABC (PK-6102), Vector / DAB / 2 (A)
M / MW 2x10 min (450w) in CB, pH 6.0 / 1:1,000 / +5°C, o/n / Dako REALTM, (K5001), Dako / DAB / 30 (P)
M / MW 20 min in CB, pH 6.0 / 1:1,000 / +4°C, o/n / Vectastain Elite ABC, (PK6100), Vector / DAB / 26 (P)
A / AC/120°C 10 min in CB, pH 6.0 / 1:2,000 / +37°C, 2 h / iVIEWTM DAB Detection, (760-091), Ventana Medical Systems / DAB / 27 (A)
A, automatic; M, manual; AC, autoclave; MW, microwave; CB, citrate buffer; FA, formic acid; o/n, overnight; RT, room temperature; A, acceptable; G, good; P, poor quality of staining 1BioGenex, San Ramon, CA; Dako (DakoCytomation), Glostrup, Denmark; Vector, Burlingame, CA; Ventana Medical Systems, Tucson, AZ; Zymed, South San Francisco, CA
Lesion / Antibody / n / Percentages of positive dichotomised assessment results within cores1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15
Pathol
IR / UBQ / 11 / A / A / 91 / 82 / A / A / A / A / A / 91 / A / A / A / A / 91
p62 / 9 / 89 / 67 / A / 88 / A / A / A / A / A / A / A / A / A / A / A
NCIs / UBQ / 11 / 82 / 73 / 36 / 18 / 82 / A / A / A / A / 64 / A / 55 / 27 / A / 80
p62 / 9 / 44 / 44 / 25 / 13 / 78 / 89 / 89 / A / A / A / A / 89 / 78 / A / A
NIIs / UBQ / 11 / 46 / 9 / 0 / 0 / 36 / 55 / 73 / 46 / 46 / 9 / 70 / 18 / 0 / 64 / 18
p62 / 9 / 11 / 11 / 0 / 0 / 22 / 11 / 67 / 56 / 78 / 11 / 56 / 33 / 22 / 56 / 33
NTs / UBQ / 11 / 82 / 82 / 82 / 82 / 91 / 91 / A / A / A / 82 / A / A / A / A / 73
p62 / 9 / 78 / 56 / A / 75 / A / 89 / 89 / 89 / 56 / A / A / A / A / A / A
NTs/
THIN / UBQ / 11 / 82 / 73 / 73 / 82 / 82 / 91 / A / A / A / 73 / A / A / A / A / 64
p62 / 9 / 67 / 56 / A / 75 / A / 89 / A / 89 / 56 / A / A / A / A / A / A
NTs/
THICK / UBQ / 11 / 18 / 9 / 27 / 9 / 46 / 73 / 91 / 73 / 36 / 36 / 40 / 91 / 94 / 73 / 36
p62 / 9 / 33 / 0 / 38 / 0 / 67 / 44 / 78 / 33 / 22 / 67 / 75 / 89 / 89 / 44 / 89
Supplement Table 3. Comparisons of dichotomised assessments of ubiquitin- and p62-immunoreactive
lesions in 15 core samples where the staining was performed applying screening antibodies,
(Trial 1) and when only the stainings with good/acceptable quality are included
Pathol IR, pathological immunoreactivity in general; NCIs, neuronal cytoplasmic inclusions; NIIs, neuronal nuclear inclusions; NTs, neurites; UBQ, ubiquitin; n, number of assessments; A, full agreement, i.e. all assessors have observed positive lesions in the core sample
Supplement Table 4. Comparisons of semiquantitative assessments of ubiquitin-, and p62-immunoreactive neuronal cytoplasmic
inclusions (NCIs)1 and neurites (NTs)2 in 15 core samples in Trial 1 when only TMAs with good/acceptable staining quality were
included
Range, Most frequent score and Percentage of most frequent score within coresNCIs / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15
UBQ (n = 11)
Range / 0-2 / 0-2 / 0-1 / 0-1 / 0-2 / 1-2 / 1-3 / 2-3 / 2-3 / 0-3 / 1-3 / 0-3 / 0-1 / 1-3 / 0-3
Most frequent score / 1 / 1 / 0 / 0 / 1 / 1 / 1,2 / 3 / 3 / 0,1 / 2 / 0 / 0 / 2 / 1
Most frequent score (%) / 73 / 64 / 64 / 82 / 73 / 91 / 46 / 55 / 55 / 36 / 50 / 55 / 80 / 55 / 50
p62 (n = 9)
Range / 0-1 / 0-1 / 0-1 / 0-1 / 0-2 / 0-2 / 0-3 / 1-3 / 1-3 / 1-2 / 1-3 / 0-1 / 0-1 / 2-3 / 1-3
Most frequent score / 0 / 0 / 0 / 0 / 1 / 1 / 1 / 2 / 1,2,3 / 1 / 3 / 1 / 1 / 3 / 2
Most frequent score (%) / 67 / 56 / 75 / 88 / 56 / 78 / 56 / 67 / 33 / 89 / 56 / 89 / 67 / 67 / 56
NTs / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15
UBQ (n = 11)
Range / 0-3 / 0-2 / 0-2 / 0-2 / 0-3 / 0-3 / 1-3 / 1-2 / 1-2 / 0-2 / 1-2 / 2-3 / 2-3 / 1-3 / 0-2
Most frequent score / 1 / 1 / 1 / 1 / 1 / 1 / 2,3 / 1 / 1 / 1 / 1 / 2 / 2 / 2 / 1
Most frequent score (%) / 55 / 64 / 73 / 73 / 55 / 64 / 46 / 55 / 91 / 64 / 80 / 55 / 73 / 45 / 64
p62 (n = 9)
Range / 0-1 / 0-1 / 1-2 / 0-1 / 1-2 / 0-3 / 1-3 / 0-1 / 0-2 / 1-2 / 1-3 / 2-3 / 1-3 / 1-3 / 1-3
Most frequent score / 1 / 1 / 1 / 1 / 2 / 1 / 3 / 1 / 0,1 / 1 / 1,2 / 3 / 2 / 1 / 1,3
Most frequent score (%) / 78 / 56 / 88 / 75 / 56 / 67 / 56 / 89 / 44 / 67 / 44 / 67 / 56 / 67 / 44
UBQ, ubiquitin;1,20 - none; 1 - some; 2 - moderate; 3 - numerous