Low background mould-prepared gelatine standards for reproducible quantification in elemental bio-imaging

Abstract

Raw intensities obtained from the ablation of mould-prepared gelatine.

Assessing the reproducibility of labelled antibody binding in quantitative multiplexed immuno-mass spectrometry imaging

“…Quantification of the lanthanides as a proxy of the antibodies was performed via external calibration using gelatine standards prepared and characterized according to a previously validated method [22]. The gelatine standards were run daily at the start of each analysis, with correlation coefficients greater than 0.99 obtained for all elements.…”

“…External calibration standards were prepared according to a previously validated protocol [22]. Briefly, a multi-elemental mix was prepared in a buffer containing 100 mM Tris-HCl buffer (pH 7.4), 10 mM EDTA, and 1% w/w polyethylene glycol in ultra-purified water (18.2 MΩ cm −1 at 25 °C, Arium Pro Vf, Sartorius, Goettingen, Germany).…”

Matching sensitivity to abundance: high resolution immuno-mass spectrometry imaging of lanthanide labels and endogenous elements in the murine brain

“…The method was developed and optimised for the calculation of SNRs using homogenous laboratory-made matrix-matched standards manufactured according to the protocol by Westerhausen et al 24 . Briefly, 100 mg high purity gelatin from bovine skin (Sigma Aldrich) was mixed with 900 µL of 172 Yb, Mn, Cu, Na and P diluted in 100 mM Tris-HCl buffer (pH 7.4), 10 mM EDTA and 1% w/w polyethylene glycol (Mn 400) prepared in Milli-Q water (18.2 MΩ cm−1 at 25 °C, Arium Pro Vf, Sartorius, Goettingen, Germany).…”

“…Briefly, 100 mg high purity gelatin from bovine skin (Sigma Aldrich) was mixed with 900 µL of 172 Yb, Mn, Cu, Na and P diluted in 100 mM Tris-HCl buffer (pH 7.4), 10 mM EDTA and 1% w/w polyethylene glycol (Mn 400) prepared in Milli-Q water (18.2 MΩ cm−1 at 25 °C, Arium Pro Vf, Sartorius, Goettingen, Germany). The spiked gelatin was heated to 55°C and filled into wells (HybriWell TM , 50 µL, 9.8 mm x 20 mm x 0.25 mm) which produced a thin gelatin film with a defined thickness (25 µm), homogeneous metal distribution and a smooth topography 24 .…”

“…Up to 32 different proteins may be labelled with different metal tags and detected with high sensitivity and subcellular lateral resolution (1 µm) 22 . Commercially available kits provide straight forward labelling and staining procedures 21 , and different standardisation approaches contribute to robust quantification and the mitigation of instrumental signal drifts 23,24 . Spatial resolution is limited by the sensitivity of the ICP-MS method which is proportional to the square of the laser beam spot radius, i.e.…”