May 06, 2025

Public workspaceStaining protocol for Imaging Mass Cytometry

DOI
dx.doi.org/10.17504/protocols.io.bp2l6ywn5vqe/v1
  • Vijayakumar Kakade1,
  • Tifanny Budiman1,
  • Lloyd Cantley1
  • 1Yale University School of Medicine
  • KPMP
    Tech. support email: info@kpmp.org
Vijayakumar R Kakade
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6ywn5vqe/v1
Protocol Citation: Vijayakumar Kakade, Tifanny Budiman, Lloyd Cantley 2025. Staining protocol for Imaging Mass Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6ywn5vqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 06, 2025
Last Modified: May 06, 2025
Protocol Integer ID: 217796
Keywords: protocol for imaging mass cytometry, imaging mass cytometry, mass cytometer, mass spectrometry, staining procedure for imc, identifying specific antigen, primary antibody, conjugated primary antibody, staining protocol, specific antigen, staining procedure, resolution laser, imaging
Funders Acknowledgements:
NIDDK
Grant ID: U01DK133768
Abstract
In Imaging Mass Cytometry (IMC), a high-resolution laser is combined with a mass cytometer that permits mass spectrometry-based, spatially reserved high-dimensional analysis of intact formalin-fixed paraffin-embedded (FFPE) tissues. The protocol summarizes the staining procedure for IMC using a cocktail of heavy metal conjugated primary antibodies identifying specific antigens.
Guidelines
Protocol Quality Control metrics
  • Antibody validation: All primary antibodies included in the panel are validated either by the vendor or internal validation. Strategies considered for antibody validation include: 1) knockout/knockdown of gene of interest, 2) independent antibody verification, 3) morphological validation by performing IF using single common protocol on human kidney and positive control tissue (lymph node, tumors etc), 4) morphological validation by a renal pathologist blinded to the antibody used and 5) counterstaining for additional markers of the same cell type.  
  • Aperio scanned image of PAS-stained section of the entire biopsy is evaluated for tissue morphology and integrity. 
  • Use 5 µm thick sections for staining. For better results, stain and ablate the tissue within 2 weeks of sectioning. 
  • Entire biopsy is ablated with increments in approximately 3 mm2 area to complete ablation of the cortical and medullary regions. Regions with artifactual section damage are excluded. 
  • Each IMC analysis includes a section from a reference kidney, both cortex and medulla (quality control), on the same slide as an internal standard. 
  • IMC is performed on the Hyperion XTi imaging system. Prior to ablation of tissue, the machine undergoes routine tuning and calibration for mass spectrometric detection of all the heavy metals used in the antibody panel each time. 
  • For each IMC ablation, the steps of antigen retrieval and cocktail hybridization will be confirmed by visual inspection of the resident cell type markers using mcd viewer to identify the nuclei, proximal tubules, glomeruli, thick ascending limb, distal convoluted tubules and collecting duct to confirm that the correct region was ablated and analyzed.  
Materials
Xylenes - JT Baker (9490-05) 
Ethanol – Decon Labs (2805M) 
10X Phosphate buffered saline (PBS) - Gibco (70011044)  
10X Tris buffered saline (TBS) - BioRad (1706435) 
Triton X-100 - Sigma (X100RS-5G)  
Epitope Retrieval Reagent pH 9 (10x) - Leica Biosystems (RE7113-CE) 
Bovine serum albumin (BSA) - American Bio (AB0048) 
Double distilled water  
PAP pen - Abcam (ab2160) 
Maxpar IMC Cell Segmentation - Standard Biotools (201500) 
Intercalator-Ir - Standard Biotools (201192A) 
Antibodies (as listed on Table 1) 
Troubleshooting
Before start

TypeTarget Cell Type Species Vendor (Cat No.) Dilution Metal Conjugate
Resident Cell PanelBeta cateninTubular epitheliumMouseStandard Biotools (3147005A) 1:500 147Sm
Aqp1Proximal tubule RabbitAbcam (ab178352-1001)1:2500173Yb
MegalinProximal tubule MouseMillipore (MABS489)1:250174Yb
UromodulinThick ascending limbRatR&D Systems (MAB5175)1:1600151Eu
CalbindinDistal convoluted tubuleMouseThermoFischer (MA524135)1:400142Nd
CK7Collecting ductMouseStandard BIotools (3164028D)1:150164Dy
NestinPodocytesMouseAbcam (ab6320-1001)1:200146Nd
VimentinFibroblasts, pericytes, podocytesMouseAbcam (ab8978-1001)1:400150Nd
CD31EndotheliumMouseAbcam (ab212712-1001)1:100149Sm
ERGEndotheliumRabbitAbcam (ab214796-1001)1:500166Er
alpha-SMASmooth muscle, mesengialMouseStandard Biotools (3141017D)1:1000141Pr
WT1PodocytesMouseThermoFischer (MA146028)1:100176Yb
Aqp2Collecting ductRabbit Abcam (ab230170)1:200154Sm
Immune Cell PanelCD68MacrophagesMouseFluidigm (3159035D)1:800159Tb
CD14Pro-Inflammatory Macrophages (M1)RabbitFluidigm (3144025D)1:100144Nd
CD163Alternative Activated Macrophages (M2)MouseBio-Rad (MCA1853)1:100148Nd
CD206Alternative Activated Macrophages (M2)RabbitAbcam (AB64693)1:400163Dy
CD11cDendritic cellRabbitAbcam (AB216655)1:200167Er
CD3T cellMouseNovus Biologicals (NBP2-54392-100)1:250170Er
CD4Helper T cellsRabbitFluidigm (3156033D)1:100156Gd
CD8aCytotoxic T cellsMouseFluidigm (3162034D)1:300162Dy
CD20B cellsMouseFluidigm (3161029D)1:150161Dy
MBPEosinophilsMouseNovus Biologicals (NBP1-42140-MTO)1:20143Nd
MPONeutrophilsRabbitAbcam (AB236022)1:500172Yb
ChymaseMast cellsRabbitAbcam (AB233729)1:400165Ho
CD56NK cellsMouseCell Signaling (97174SF)1:200175Lu
Injury Cell PanelKim1Epithelial injury/repairMouseR&D Systems (MAB1750-100)1:300160Gd
Ki67ProliferationMouseFluidigm (3168022D)1:100168Er
IL9CytokineRabbitAbcam (ab181397)1:250153Eu
FACL4FerroptosisRabbitAbcam (ab240135)1:400155Gd
MCP-1CytokineMouseNovus Biologicals (NBP2-22115)1:300169Tm
TNFaCytokineRabbitAbcam (ab271989)1:300145Nd
LC3bAutophagyRabbitAbcam (ab221794-1001)1:200158Gd
VCAM-1Epithelial injury/repaitRabbit Abcam (ab271899-1001)1:200152Sm
Segmentation KitICSK1Membrane-Standard Biotools (201500)1:400195Pt
ICSK2Membrane-Standard Biotools (201500)1:400196Pt
IntercalatorDNA-Standard Biotools (201192A)1:1000191Ir/193Ir
Table 1. Antibody Panel

Staining Tissue Sections
4h 20m
Bake the slides DurationOvernight at 60°C. Ensure that all visible wax has been removed

Dewax the slides in xylenes in the fume hood for Duration00:10:00

10m
Repeat step 2 and dewax the slides in fresh xylene in the fume hood for Duration00:10:00

10m
Hydrate the slides in descending grades of ethanol (100%, 95%, 80%, 70%) for Duration00:05:00 each.

5m
Wash the slides in double distilled water for Duration00:05:00 in a Coplin jar placed on an orbital shaker plate with gentile agitation.

5m
Perform antigen retrieval bu immersing slides in 1X epitope retrieval bugger (pH 9.0) for Duration00:20:00 in a steamer

20m
Prepare 1X epitope retrieval buffer from its 10X stock solution.
Cool slides for Duration00:40:00

40m
Wash the slides in double distilled water for Duration00:05:00 in a Coplin jar with gentle agitation on an orbital shaker. Perform this step twice.

5m
Wash the slides with 1X TBS for Duration00:10:00 with gentle agitation on an orbital shaker.

10m
Use a PAP pen to draw an outline encircling the sample.
Block with 3% BSA in 1X TBS for Duration01:00:00 at room temperature in a hydration chamber.

1h
You can use an empty pipette tip box where the slides rest on the tip shelf and the bottom is filled halfway with water.
Blocking solution should be diluted from 10% BSA freshly made from powder. The remaining 10% BSA should be aliquoted and stored at -20°C and diluted at time of use.
Use enough blocking solution to cover the section.
Prepare the antibody cocktail calculating the total volume of antibodies at the concentrations specific for the assay and bring the volume up to a final volume using 0.5% BSA. in 1X TBS.
Dilutions can be found on Table 1.
Place the slides in a hydration chamber and pipette the antibody mix on to the section.
Store the antibody cocktail mix on ice and add it on to the samples within 1-2 hours of preparation for best results.
Incubate overnight with the antibody cocktail at 4°C in a hydration chamber.
Wash the slides in 0.1% Triton X-100 in 1X TBS for Duration00:05:00 in Coplin jars with slow agitation on an orbital shaker.

5m
Repeat step 15 two more times.
Wash the slides in 1X TBS for Duration00:05:00 with gentle agitation on an orbital shaker.

5m
Repeat step 17.
Stain the tissue with Intercalator-Ir in 1X PBS (1:1000) for Duration01:00:00 at room temperature in a hydration chamber.

1h
Wash the slides in double distilled water for Duration00:05:00 with gentle agitation on an orbital shaker.

5m
Air dry the slides for at least Duration00:20:00 at room temperature.

20m
Imaging Mass Cytometry
Load the stained slide into the pre-tuned and pre-calibrated Hyperion XTi IMC System.
Create a new project on the CyTOF software.
Draw a panorama that covers the tissue that will be ablated.
Determine regions of interest (ROIs) to ablate with increments less than 3mm2 on the tissue, ensuring that cortical and medullary regions are separate within the panorama created. Regions with artificial section damage are excluded.
Create or select an antibody/metal template.
Make sure to select "generate txt file".
Save the project file and start ablation of tissue.
Image Verification
Once ablation is complete, load the file on MCD viewer to check the quality of each image and channel.