Antigens were selected based on previously published single-cell mass cytometry and single-cell RNA sequencing data on the human fetal intestinal samples (1 (link), 12 (link), 13 (link)). Antibodies used for IMC are listed in Table 1 . 16 of the 34 antibodies used in the current panel were directly purchased from Fluidigm, which were already conjugated with metals. For the remaining 18 antibodies, BSA-free and carrier-free formulations of antibodies were purchased from different suppliers and initially tested for performance by immunohistochemical staining (IHC) on human fetal intestine and tonsil. Subsequently, antibodies with an appropriate signal intensity were conjugated to lanthanide metals using the MaxPar Antibody Labeling Kit (Fluidigm) following the manufacturer's instructions. Post-conjugation, all antibodies were eluted in 100 μl W-buffer (Fluidigm) and 100 μl antibody stabilizer buffer (Candor Bioscience, Wangen im Allgäu, Germany) supplemented with 0.05% sodium azide.
W buffer
Manufactured by Standard BioTools
Sourced in Germany
W-buffer is a laboratory reagent used in various applications. It serves as a buffer solution, maintaining a specific pH range to support various biochemical processes. The core function of W-buffer is to provide a controlled chemical environment for experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Maxpar antibody labeling kit, by Standard BioTools (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Iridium dna intercalator, by Standard BioTools (1 mentions)
Cytof instrument, by Standard BioTools (1 mentions)
3 element tuning slide, by Standard BioTools (1 mentions)
Mcd viewer, by Standard BioTools (1 mentions)
4 protocols using w buffer
1
Comprehensive Intestinal Immune Profiling
2
Monoisotopic Pd Antibody Conjugation
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Two Pd isotopes, 106Pd and 110Pd, overlap with 2 Cd isotopes, 106Cd and 110Cd, having similar mass weights. Hence, three monoisotopic palladium nitrate compounds, 104Pd, 105Pd and 108Pd, were previously dissolved in HCl to 50 mM concentration to load onto DOTA and ITCBE chelators in accordance with earlier reports5 (link). Pd isotopes suspended in 5 N HCl were lyophilized overnight and suspended in nitric acid to generate Pd(NO3)2 salts. Isotopically enriched Pd nitrate solution was lyophilized overnight and suspended in water to 50 mM concentration. We utilized a similar approach for Cd-MCP9 antibody conjugation protocol to load Pd metals onto MCP9 polymers. Briefly, 13 µl of monoisotopic Pd isotope was loaded onto 200 µg of MCP polymer and washed twice with L and once with C buffer after incubation at 37 °C for one hour. Pd-loaded MCP9 polymer was then mixed with reduced CD45 antibody and incubated for 90 min at 37 °C. After incubation, antibodies were transferred to 100 kDa filter and washed using W buffer (Fluidigm, San Diego, CA). Antibody concentration was determined based on absorption at 280 nm using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA) All conjugated antibodies were then diluted to 0.5 mg/ml final concentration in HRP-protector (Candor Bioscience GmbH, Wangen, Germany).
3
Comprehensive Single-Cell Cytometry Analysis
4
Lanthanide-Conjugated Antibody Imaging
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Antibodies employed in this study were conjugated to purified lanthanide metals using the Maxpar antibody labelling kit and protocol (Fluidigm, San Francisco, CA, USA). Antibodies were eluted in 50 µl antibody stabilizer solution (Candor Bioscience, Wangen im Allgäu, Germany) supplemented with 0,05 % sodium azide and 50 µl W-buffer (Fluidigm). After conjugation, all antibodies were tested by IHC on 4 µm tonsil tissue to confirm that the labelling process did not affect antibody performance. IMC immunodetection was performed following the methodology published previously by our lab (11) using the antibodies and conditions described in supplementary table 1. Tissue sections were ablated within a week after immunodetection by using the Hyperion mass cytometry imaging system (Fluidigm). The Hyperion was autotuned using a 3-element tuning slide (Fluidigm) as described in the Hyperion imaging system user guide. An extra minimum threshold of 1500 mean duals of 175 Lu was used. Four 1000x1000 µm regions of interest per sample were selected based on haematoxylin and eosin (H&E) stains on consecutive slides and ablated at 200 Hz. Data was exported as .MCD files and .txt files and visualised using the Fluidigm MCD viewer. For downstream analysis, the .MCD files were transformed to either 32-bit multi-tiff or single-marker tiff images in the MCD viewer.
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