Each of the 8–10 Affimers, isolated for each of the three protein targets (ACTN2, Z1Z2 and ZASP) were tested for their ability to label Z-discs in heart sections, and from these, the Affimer that gave the best signal, specific Z-disc labelling with low background, was taken forward for the remaining experiments presented here. To prepare sections, frozen left ventricular heart tissue was embedded in O.C.T (optimal cutting temperature) compound and brought up to cryosection temperature of –20°C. 10 μm thick sections were cut using a cryostat (Leica Biosystems) and adhered directly to SuperFrost Plus slides (Fisher Scientific). A PAP pen was used to draw a hydrophobic barrier around the section and the tissue was fixed in 4% paraformaldehyde for 60 min at room temperature before washing three times in PBS containing Tween-20 for 5 min each.
To label sections, the sections were first blocked in in phosphate buffer (PBS) containing 0.5% Triton X-100 and 10% BSA (bovine serum albumin) for 1 h, then incubated with 10 mg/ml Affimer or primary antibody, diluted in blocking buffer, for 1 h at RT or O/N at 4°C. Following washing, in PBS -Tween, samples were either incubated for an hour with secondary antibody diluted in blocking buffer and either washed again or (for Affimer staining only, where a second incubation step is not required) mounted directly by adding a drop of ProLong Gold Antifade (Invitrogen) onto the section, then placing a cleaned glass coverslip [#1.5: (Scientific Laboratory Supplies)] on top of the samples. All the samples were labelled for identification using an alphanumeric code, to avoid bias in imaging and subsequent analysis.
Confocal imaging used an inverted Zeiss LSM880 in Airyscan mode, using the ×40 N.A 1.4 objective lens and the same laser power settings for each sample. STED imaging used an Abberior STEDYCON and a ×100, N.A. 1.4 objective lens, with the depletion laser set for ∼50nm resolution.
In addition to the Affimers, commercial mouse monoclonal anti-actinin antibody EA-53 (Sigma-Aldrich, 1:500: raised to the full length ACTN protein) and a mouse monoclonal antibody to human desmoglein-2 (CCSTEM28; eBiosciences from Thermo Scientific, 1:200) were used followed by secondary anti-mouse StarRed antibodies (Abberior, 1:100). The rabbit polyclonal titin Z1Z2 antibody was generously provided by Bang et al. (14 (link)) (raised against the NH2-terminal 195 residues of the human cardiac titin) and used with secondary anti-rabbit Star Red antibodies. Affimers used in these experiments were directly labelled with STAR 580.
To label sections, the sections were first blocked in in phosphate buffer (PBS) containing 0.5% Triton X-100 and 10% BSA (bovine serum albumin) for 1 h, then incubated with 10 mg/ml Affimer or primary antibody, diluted in blocking buffer, for 1 h at RT or O/N at 4°C. Following washing, in PBS -Tween, samples were either incubated for an hour with secondary antibody diluted in blocking buffer and either washed again or (for Affimer staining only, where a second incubation step is not required) mounted directly by adding a drop of ProLong Gold Antifade (Invitrogen) onto the section, then placing a cleaned glass coverslip [#1.5: (Scientific Laboratory Supplies)] on top of the samples. All the samples were labelled for identification using an alphanumeric code, to avoid bias in imaging and subsequent analysis.
Confocal imaging used an inverted Zeiss LSM880 in Airyscan mode, using the ×40 N.A 1.4 objective lens and the same laser power settings for each sample. STED imaging used an Abberior STEDYCON and a ×100, N.A. 1.4 objective lens, with the depletion laser set for ∼50nm resolution.
In addition to the Affimers, commercial mouse monoclonal anti-actinin antibody EA-53 (Sigma-Aldrich, 1:500: raised to the full length ACTN protein) and a mouse monoclonal antibody to human desmoglein-2 (CCSTEM28; eBiosciences from Thermo Scientific, 1:200) were used followed by secondary anti-mouse StarRed antibodies (Abberior, 1:100). The rabbit polyclonal titin Z1Z2 antibody was generously provided by Bang et al. (14 (link)) (raised against the NH2-terminal 195 residues of the human cardiac titin) and used with secondary anti-rabbit Star Red antibodies. Affimers used in these experiments were directly labelled with STAR 580.
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