The Antibody Society’s antibody validation webinar series

Voskuil, Jan; Bandrowski, Anita; Begley, C. Glenn; Bradbury, Andrew; Chalmers, Andrew D.; Gomes, Aldrin V.; Hardcastle, Travis; Lund-Johansen, Fridtjof; Plückthun, Andreas; Roncador, Giovanna; Solache, Alejandra; Taussig, Michael J.; Trimmer, James S.; Williams, Cecilia; Goodman, Simon L.

doi:10.1080/19420862.2020.1794421

“…13 e, f) [3], while shank, a PSD scaffold protein, is located to the deeper layer of the PSD (Fig. 13 g, h) [24]. Each of these antibodies were speci cally localized to their expected sites without cross-labeling other cellular elements.…”

Section: Considerations On Interpretation Of Immunogold Labeling Resultsmentioning

confidence: 98%

Optimization of Protocols for Pre-Embedding Immunogold Electron Microscopy of Neurons in Cell Cultures and Brains

Tao-Cheng

¹

,

Crocker

²

,

Moreira

³

et al. 2021

Preprint

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Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

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“…One of the crucial issues for any immunolabeling study is the specificity of the antibody used to label the protein of interest. Many different validation protocols are summarized in reviews [ 31 , 32 ], but cross-reactive antibodies still pose problems due to lack of multiple approaches in verifications.…”

Section: Resultsmentioning

confidence: 99%

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

Tao-Cheng

¹

,

Crocker

²

,

Moreira

³

et al. 2021

View full text Add to dashboard Cite

Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

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“…The reproducibility of results may be affected by parameters such as changes in the reagents, e.g. use of different lots or batches, different experimenters, and using cell lines at different passages [2,55–57]. Hence, experiments performed closely together may be expected to display greater similarity than experiments performed at more distant points in time during the decades of anti-cancer compound testing by the NCI60.…”

Section: Resultsmentioning

confidence: 99%

Data variability in standardised cell culture experiments

Reddin

¹

,

Fenton

²

,

Wass

³

et al. 2021

Preprint

0

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Despite much debate about a perceived 'reproducibility crisis' in the life sciences, it remains unclear what level of replicability is technically possible. Here, we analysed the variation among drug response data of the NCI60 project, which for decades has tested anti-cancer agents in a 60-cell line panel following a standardised protocol. In total, 2.8 million compound/cell line experiments are available in the NCI60 resource CellMiner. The largest fold change between the lowest and highest GI50 (concentration that reduces cell viability by 50%) in a compound/cell line combination was 3.16 x 1010. All compound/ cell line combinations with >100 experiments displayed maximum GI50 fold changes >5, 99.7% maximum fold changes >10, 87.3% maximum fold changes >100, and 70.5% maximum fold changes >1000. FDA-approved drugs and experimental agents displayed similar variation. The variability remained very high after removal of outliers and among experiments performed in the same month. Hence, our analysis shows that high variability is an intrinsic feature of experimentation in biological systems, even among highly standardised experiments in a world-leading research environment. Thus, a narrow focus on experiment standardisation does not ensure a high level of replicability on its own.

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The Antibody Society’s antibody validation webinar series

Cited by 37 publications

References 35 publications

Optimization of Protocols for Pre-Embedding Immunogold Electron Microscopy of Neurons in Cell Cultures and Brains

Optimization of Protocols for Pre-Embedding Immunogold Electron Microscopy of Neurons in Cell Cultures and Brains

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

Data variability in standardised cell culture experiments

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