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: February 28, 2022
Last Modified: February 28, 2022
Protocol Integer ID: 58861
Keywords: lanthanides-tagged antibody, immunostaining, imaging mass spectrometry, secondary ion mass spectrometry, C60-SIMS
Funders Acknowledgement:
NIH
Grant ID: UG3CA256962
Abstract
Imaging mass spectrometry (IMS) is able to image the multiple lanthanides-tagged antibodies (up to 40) simultaneously at a subcellular resolution on a single tissue section. This protocol is developed for optimized immunostaining of frozen-hydrated tissue with antibodies cocktail (10-25 Abs), which produces consistent antibody affinity and improved IMS image quality using C60-secondary ion mass spectrometry (C60-SIMS).
Materials
• Five micrometer fresh snap-frozen human liver sections on Au-coated Silicon wafer (2.2cm*2.2cm)
• 1% Paraformaldehyde (diluted from 16% solution from Thermo Fisher Scientific)
• Methanol (Sigma)
• 3% Goat serum (diluted from 10% stock from Thermo Fisher Scientific)
• DPBS (Corning)
• Wash buffer [DPBS supplemented with 0.05% Tween(Thermo Fisher Scientific) and 1% BSA(Sigma)]
• Metal-conjugated antibodies as in Table 1 below. Please refer to Fluidigm Maxpar Antibody Labeling User Guide Chapter 3 for detailed conjugation steps. Maxpar® X8 Antibody Labeling Kit with all the metal tags in Table 1 are purchased from Fluidigm
Table 1 Antibody panel for human liver tissue
A
B
C
D
E
Metal
Target
Clone/Host
Cell/Pathway
Manufacturer/Catalog No.
89Y
CD45
D9M8I, Rabbit IgG
Pan leukocyte
Cell Signaling/13917S
113 In
CD4
RPA-T4, mouse monoclonal
T cell
Novus/NBP2-25199
141Pr
SMA
1A4, Mouse IgG2a
Vascular walls/Hepatic
Stellate cells/fibroblasts
Fluidigm/3141017D
143Nd
GFAP
EPR1034Y, Rabbit monoclonal
Ito Stellate Cells
Abcam/ab218309
145Nd
Heppar-1
HepPar1, mouse monoclonal
hepatocytes
Novus/NBP3-08970
147Sm
Glul
ab240193,Rabbit monoclonal
Pericentral hepatocytes (Zone 3)
Abcam/ab240193
148Nd
CD31
JC/70A, Mouse monoclonal
Endothelial cells
Abcam/ab264090
151Eu
CD68
D4B9C,Rabbit IgG
Macrophages
Cell Signaling/76437S
153Eu
CD32
FUN-2, Mouse IgG2b
Macrophages
Fluidigm/3153018B
158Gd
Arginase1
D4E3M™,Rabbit IgG
Zone 1-2 hepatocytes
Cell
Signaling/93668S
161Dy
Albumin
EPR20195, Rabbit
monoclonal
Periportal (Zone 1)
Abcam/Ab271979
166Er
CK19
SPM561, Mouse monoclonal
Cholangiocytes
(Portal triad)
Abcam/ab212569
168Er
Ki-67
Ki-67, Mouse IgG1
Proliferation
(Midlobular)
Cell Signaling/9449S
169Tm
CD34
QBEND/10, Mouse / IgG1
Endothelial cells
Abcam/ ab198395
170 Er
EpCAM
E6V8Y, Rabbit IgG
Hepatic stem/progenitor cells
Cell Signaling /93790S
171Yb
LYVE1
EPR21857, Rabbit monoclonal
Sinusoidal endothelial cells
Abcam/ab232935
176Yb
EGFR
EP38Y, Rabbit monoclonal
cell membrane
Abcam/ ab272293
176Yb
Na/K ATPase
D4Y7E, Rabbit IgG
cell membrane
Cell Signaling/ 23565S
191Ir
Nuclear DNA
Nuclei
Fluidigm/ 201192B
196Pt
Human collagen I
EPR7785,
Rabbit monoclonal
Collagen
Abcam/ab215969
• Intercalator-Ir (500µM, Fluidigm)
• DI water (Sigma)
• Water-Repellent Slides Marker Pen (Dando Sangyo Co Ltd. Japan)
• glass petri dish (diam 40mm, Sigma)
Safety warnings
Wear proper PPE when handling the agents. Discard the waste in the biohazard waste container
Cut the fresh frozen sections at 5µm using a cyromicrotome and mount them on Au-coated silicon slides
Place the slides at -20 ◦C for 1h and then at 4 ◦C for 30 min for temperature equilibrium
Fix the tissue slides in 1% PFA for 5 min at 4 ◦C, then in prechilled methanol for 5 min at -20 ◦C
Wash the slides twice for 5 min in a glass petri dish by submerging the slides in 25 ml wash buffer, following by rehydrating the slides for 5 min in a glass container with 25ml DPBS and rinsing the slides for 5 min in a glass container with 25ml wash buffer
Use the Liquid-Repellent Slide Marker Pen to draw a circle around the tissue section to create a barrier to contain the solutions on the tissue sections
Apply 100 µl 3% goat serum solution to each slide for 1 h at RT and then remove excess block solution by tapping on a tissue
Prepare the antibody cocktail. Vortex all the antibodies for 15 s and then centrifuge at 12,000G for 2 min, followed by diluting the upper liquid of each antibody in 0.5% PBS as desired concentration
Add 100 µl of the antibody cocktail to each section and incubate overnight at 4◦C in a sealed glass petri dish in a fridge
After the incubation, wash the sections two times for 5min in a glass petri dish with 5 ml wash buffer with slow agitation
Incubate the slides with 100 µl 1:100 dilution of Intercalator-Ir in DPBS for 30 min at RT
After nuclei staining, wash the slides for 5 min in a glass container with 5 ml DPBS
Quickly dip the slides in a container with 5 ml DI water for 15 s, repeat three times