From CLARITY Wiki
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|Murray et al, Cell 2015; Liu et al, Neuropath. Appl. Neurobiol. 2016; Lee et al, Scientific Reports 2016 |
|Murray et al, Cell 2015; Liu et al, Neuropath. Appl. Neurobiol. 2016; Lee et al, Scientific Reports 2016 |
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+ | |Keratin-5 |
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+ | |1:100 |
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+ | |Mouse mammary gland |
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+ | |1 |
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+ | |BioLegend |
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+ | |905501 |
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+ | |Lloyd-Lewis et al, Breast Cancer Res. 2016 |
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+ | |- |
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+ | |Ki67 |
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+ | |1:100 |
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+ | |Mouse brain |
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+ | |1 |
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+ | |Abcam |
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+ | |ab16667 |
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+ | |Murray et al, Cell 2015 |
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+ | |- |
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+ | |rowspan="3"|Laminin |
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+ | |1:100-1:500 |
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+ | |Mouse brain; mouse liver; mouse skeletal muscle |
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+ | |1 |
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+ | |Sigma |
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+ | |L9393 |
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+ | |Lee et al, Scientific Reports 2016; Verma et al, Skeletal Muscle Regen. in the Mouse 2016 |
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+ | |- |
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+ | |rowspan="2"|1:100 |
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+ | |Mouse skeletal muscle |
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+ | |Sigma |
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+ | |L0663 |
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+ | |Verma et al, Skeletal Muscle Regen. in the Mouse 2016 |
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+ | |- |
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+ | |Mouse pancreas; human pancreas |
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+ | |1+ |
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+ | |Millipore |
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+ | |ab2034 |
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+ | |Hsueh et al, Scientific Reports 2017 |
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+ | |- |
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+ | |Lectin |
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+ | |1:50 |
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+ | |Mouse skeletal muscle |
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+ | |Vector Labs |
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+ | |DL-1174, DL-1177 |
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+ | |Verma et al, Skeletal Muscle Regen. in the Mouse 2016 |
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Revision as of 22:47, 10 January 2018
Staining with either antibodies or dyes is performed following sample clearing and a two day PBST buffer wash. Afterwards, sample imaging using three-dimensional techniques, such as confocal microscopy, can be performed. Tissue samples can then be stored in PBST or processed for additional rounds of immunostaining and imaging.
Contents |
Guidelines for immunostaining
Successful immunostaining results are often strongly dependent on the antibody choice. Protocol modifications and optimizations may need to be performed for different antibodies, and so one standard procedure can not be applied. The following are guidelines for obtaining successful staining results.
Staining parameters
- Use long enough incubation times - Multiple days for incubation will likely be needed. For a 1 mm section, a good starting point is: 2 day incubation in primary antibody, 1 day wash with buffer, 2 day incubation in secondary antibody, 1 day wash with buffer.
- Use high antibody concentrations - Start with antibody concentrations in the range of 10 μg/mL (1:100) to 20 μg/mL (1:50), and lower as needed.
- Use high temperature - Incubating at 37°C will promote quicker antibody penetration into the clarified tissue sample. If antibody binding is a problem, though, incubation at lower temperatures (4°C, for example) can be done for longer times. A combination of temperatures (37°C followed by 4°C) may combine the benefits of both.
- PBST buffer - While the sample can be transferred to other buffers for immunostaining, PBST buffer is known to work well.
Other recommendations
- Passive clearing - Immunostaining seems to work best in samples that have been passively cleared as opposed to ETC. It is recommended to use slightly longer passive clearing incubation times to get the sample to near transparency even in the clearing solution.
- Smaller sections - 1-2 mm thick sections will provide the best results. Whole tissue staining will need signficantly longer incubation times and antibodies may still not be able to fully penetrate to the core of the sample (a whole mouse brain, for example, took 2 weeks incubation in each antibody with 1 week long buffer wash steps).
- Blocking agent - Use of a blocking agent in brain tissue has not shown an obvious difference in immunostaining results and is probably not needed. This may not be the case for other tissue types.
- Hydrogel solution modification - The hydrogel solution can be modified to reduce the amount of crosslinking in the hydrogel-embedded tissue and therefore allow faster and better penetration of antibodies during staining.
Recommended antibodies and dyes
The following is a list of antibodies and dyes that should provide reliable staining results. These recommendations may be useful to start with when testing out the CLARITY method for the first time.
- Parvalbumin (abcam)
- Tyrosine Hydroxylase (abcam)
- Propidium Iodide
- DAPI (may wash out in mounting solution after 1-2 days)
- Neurofilament NF-H (chicken antibody from Aves)
Primary antibodies used in CLARITY literature
The following table details primary antibodies that have been used on CLARITY or PACT (passive CLARITY technique) tissue samples in the published literature. It is meant to provide guidance for those unsure of which antibodies may provide good immunostaining results in their CLARITY-processed samples. For more information on how a specific antibody was used and the results obtained, check the corresponding reference (listed in the Journal Articles section).
Antigen | Dilution (vol. antibody soln : vol. buffer) |
Tissue type | Tissue thickness (mm) | Vendor | Catalog number | References |
---|---|---|---|---|---|---|
2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) | 1:300 | Mouse brain | 1 | Millipore | MAB326 | Lee et al, Scientific Reports 2016; Saul et al, Genome Research 2017 |
Sigma | c5922 | Lee et al, Scientific Reports 2016 | ||||
Acetylated tubulin | 1:500 | Mouse brain; mouse testis; mouse lung | 1 | Sigma | T7451 | Lee et al, Scientific Reports 2016 |
Active caspase-3 | 1:500 | Mouse brain | 1 | Cell Signaling Technology | 9661 | Lee et al, Scientific Reports 2016 |
Alpha-adducin | 1:200 | Abcam | ab51130 | Treweek et al, Nature Protocols 2015 | ||
Alpha-smooth muscle actin | 1:50 | Mouse pancreas | 1+ | Abcam | ab5694 | Lee et al, BMC Develop. Bio. 2014 |
1:200-1:300 | Mouse mammary gland | 1 | Abcam | ab5694 | Lloyd-Lewis et al, Breast Cancer Res. 2016 | |
Alpha-synuclein | 1:50 | Human brain | 3 | Becton Dickinson | 610787 | Liu et al, Neuropathol. Appl. Neurobiol. 2016 |
Anti-Mullerian hormone (AMH) | 1:50 | Mouse ovary | Santa Cruz Biotechnology | sc-6886 | Feng et al, Scientific Reports 2017 | |
Aquaporin-4 (AQP4) | 1:100 | Mouse intestine | Santa Cruz Biotechnology | sc-20812 | Neckel et al, Scientific Reports 2016 | |
Aromatase (CYP19) | 1:50 | Mouse ovary | Abcam | ab35604 | Feng et al, Scientific Reports 2017 | |
B-cell lymphoma-2 (Bcl-2) | 1:250 | Mouse brain | 1 | Santa Cruz Biotechnology | sc-7382 | Lee et al, Scientific Reports 2016 |
Beta-amyloid | 1:100 | Mouse brain | 1 | Cell Signaling Technology | 2454 | Murray et al, Cell 2015 |
Beta-amyloid (A beta) 4G8 | Human brain | 0.5 | Covance | Ando et al, Acta Neuropath. 2014 | ||
Beta-catenin | 1:500 | Mouse brain | 1 | Becton Dickinson | 610153 | Lee et al, Scientific Reports 2016 |
Beta-spectrin II | 1:200 | BD Biosciences | 612563 | Treweek et al, Nature Protocols 2015 | ||
Beta-tubulin | 1:200 | Santa Cruz Biotechnology | sc-9104 | Treweek et al, Nature Protocols 2015 | ||
Beta-tubulin III (Tuj1) | 1:350 | Mouse brain | 1 | Covance | MMS-435P | Lee et al, Scientific Reports 2016 |
1:100-1:300 | Mouse brain; mouse spinal cord | 1 | Sigma | T8660, T2200 | Lee et al, Scientific Reports 2016; Lai et al, PLoS One 2016 | |
1:400 | Mouse intestine; human intestine | Covance | PRB-435P | Neckel et al, Scientific Reports 2016 | ||
1:1000 | Organoid | Promega | G712A | Lee et al, Neuropsychopharmacology 2017 | ||
1:50 | Mouse ovary | Abcam | ab18207 | Feng et al, Scientific Reports 2017 | ||
1:100 | Mouse pancreas; human pancreas | 1+ | Millipore | MAB1637 | Hsueh et al, Scientific Reports 2017 | |
BH interacting domain death agonist (BID) | 1:25 | Mouse ovary | Santa Cruz Biotechnology | sc-292494 | Feng et al, Scientific Reports 2017 | |
Brain-derived neurotropic factor (BDNF) | 1:50 | Mouse ovary | Abcam | ab72439 | Feng et al, Scientific Reports 2017 | |
c-Fos | 1:500 | Mouse brain | 1 | Santa Cruz Biotechnology | sc-253 | Lee et al, Scientific Reports 2016 |
C-Kit | 1:200 | Mouse intestine | Dako | A4502 | Neckel et al, Scientific Reports 2016 | |
Calbindin (CB) | 1:500 | Mouse brain | 0.5 | Swant | CB-38 | Unal et al, J. Neurosci. 2015 |
1:100 | Mouse brain | 1 | Cell Signaling Technology | 13176 | Murray et al, Cell 2015 | |
Abcam | ab11426 | Murray et al, Cell 2015 | ||||
Calbindin D-28k | 1:100-1:500 | Mouse brain | 0.25-1 | Swant | 300 | Lee et al, Scientific Reports 2016; Phillips et al, Scientific Reports 2016 |
Calcitonin gene-related peptide (CGRP) | 1:300 | Mouse brain | 1 | Abcam | ab36001 | Lee et al, Scientific Reports 2016 |
Calretinin | 1:100 | Mouse brain | 0.5 | Swant | 7699/3H | Unal et al, J. Neurosci. 2015 |
1:500 | Mouse brain | 1 | Swant | 7697 | Lee et al, Scientific Reports 2016 | |
1:800 | Mouse brain | 1 | Millipore | AB5054 | Lee et al, Scientific Reports 2016 | |
Caveolin | 1:300 | Mouse brain | 1 | Abcam | ab18199 | Lee et al, Scientific Reports 2016 |
CD 31 | 1:50-1:100 | Mouse intestine; mouse pancreas; human pancreas | 1+ | Abcam | ab28364 | Neckel et al, Scientific Reports 2016; Hsueh et al, Scientific Reports 2017 |
1:100 | Mouse spinal cord; mouse embryo; rat brain | 4 | Santa Cruz Biotechnology | Woo et al, Exp. Mol. Med. 2016 | ||
1:500 | Mouse brain | 1 | Becton Dickinson | 557355 | Lee et al, Scientific Reports 2016 | |
CD133 | 1:500 | Mouse brain | 1 | Millipore | MAB4310 | Lee et al, Scientific Reports 2016 |
CD34 | 1:100 | Mouse spleen | Santa Cruz Biotechnology | sc-9095 | Epp et al, eNeuro 2015 | |
CD8-PE | 1:100 | Mouse colon; mouse reproductive tract | BioLegend | Kieffer et al, eLife 2017 | ||
Choline acetyltransferase (ChAT) | 1:50-1:200 | Mouse brain | 0.5-1 | Millipore | AB144P | Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Unal et al, J. Neurosci. 2015; Lee et al, Scientific Reports 2016 |
1:100 | Mouse intestine | Chemicon International | AB144P | Neckel et al, Scientific Reports 2016 | ||
Collagen type III | 1:300 | Mouse brain | 1 | Abcam | ab7778 | Lee et al, Scientific Reports 2016 |
Collagen type IV | 1:300 | Mouse brain; mouse kidney; human spinal cord | 1 | Abcam | ab6586 | Lee et al, Scientific Reports 2016 |
1:100 | Mouse lung | Millipore | AB756P | Epp et al, eNeuro 2015 | ||
Mouse pancreas; human pancreas | 1+ | Millipore | MAB8201 | Hsueh et al, Scientific Reports 2017 | ||
Collagen type IX | 1:20 | Chicken embryo foot | DSHB | 2C2 | Botelho et al, J. Exp. Zool. 2017 | |
Complex IV subunit I (COX1) | 1:100 | Mouse brain; human brain | 0.25 | Abcam | ab14705 | Phillips et al, Scientific Reports 2016 |
Complex IV subunit IV (COX4) | 1:100 | Mouse brain; human brain | 0.25 | Abcam | ab14744 | Phillips et al, Scientific Reports 2016 |
Ctip2 | 1:800 | Mouse brain | 1 | Abcam | ab18465 | Lee et al, Scientific Reports 2016 |
Cut-like homeobox-1 (Cux1) | 1:150 | Mouse brain | 1 | Santa Cruz Biotechnology | sc-13024 | Lee et al, Scientific Reports 2016 |
Cytokeratin | 1:400 | Mouse intestine | Dako | Z0622 | Neckel et al, Scientific Reports 2016 | |
Cytokeratin-19 (CK19) | 1:100 | Mouse liver | 1+ | Developmental Studies Hybridoma Bank | Troma-III | Font-Burgada et al, Cell 2015 |
Cytokeratin-20 (CK20) | 1:100 | Mouse pancreas; human pancreas | 1+ | Dako | Ks20.8 | Hsueh et al, Scientific Reports 2017 |
Cytokeratin-7 (CK7) | 1:100 | Mouse pancreas; human pancreas | 1+ | Dako | OV-TL 12/30 | Hsueh et al, Scientific Reports 2017 |
Cytokeratin-8 | 1:50-1:200 | Mouse mammary gland | 1 | Developmental Studies Hybridoma Bank | Troma-I | Lloyd-Lewis et al, Breast Cancer Res. 2016 |
Digoxigenin (DIG) | 1:500 | Mouse brain | 1 | Sylwestrak et al, Cell 2016 | ||
Dopamine transporter (DAT) | 1:500 | Mouse brain | 0.3 | Millipore | MAB369 | Stout et al, ACS Chem Neuro 2016 |
Dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) | 1:1000 | Mouse brain | 1 | Cell Signaling Technology | 19A3 | Poguzhelskaya et al, Mol. Neurodeg. 2014 |
Doublecortin (DCX) | 1:500 | Mouse brain | 1 | Santa Cruz Biotechnology | sc-8066 | Lee et al, Scientific Reports 2016 |
DsRed | 1:300-1:500 | Mouse brain; Xenopus brain; Zebrafish | 3 | Clontech Laboratories | 632496 | Plummer et al, Development 2015; Fini et al, Scientific Reports 2017; Passoni et al, Disease Models and Mech. 2017 |
Dystrophin | 1:100 | Mouse skeletal muscle | Abcam | ab15277 | Verma et al, Skeletal Muscle Reg. in the Mouse 2016 | |
E-Cadherin | 1:50-1:200 | Mouse mammary gland; mouse embryonic epididymis; mouse embryonic kidney; mouse embryonic lung | 1 | Cell Signaling Technology | 3195 | Lloyd-Lewis et al, Breast Cancer Res. 2016; Hirashima and Adachi, PLoS One 2015 |
1:50-1:300 | Mouse mammary gland; Zebrafish; rat kidney | 1-1.5 | Becton Dickinson | 610182 | Lloyd-Lewis et al, Breast Cancer Res. 2016; Cronan et al, Immunity 2016; Unnersjo-Jess et al, Kidney International 2016 | |
Epidermal growth factor receptor (EGFR) | 1:500 | Mouse brain | 1 | Abcam | ab2430 | Lee et al, Scientific Reports 2016 |
ER81 | 1:100 | Mouse brain | 1 | Covance | PRB-362C | Murray et al, Cell 2015 |
Estrogen related receptor alpha (ERR-alpha) | Mouse brain | 0.2 | Santa Cruz Biotechnology | sc-66882 | Saul et al, Genome Research 2017 | |
Excitatory amino acid transporter-2 (EAAT2) | 1:100 | Mouse brain | 1 | Abcam | ab178401 | Murray et al, Cell 2015 |
Forkhead box protein P2 (FOXP2) | 1:100 | Mouse brain | 1 | Abcam | ab1307 | Murray et al, Cell 2015 |
Gamma-aminobutyric acid (GABA) | 1:500 | Mouse brain | 1 | Sigma | A2052 | Lee et al, Scientific Reports 2016 |
Glial fibrillary acidic protein (GFAP) | 1:50-1:200 | Mouse brain; human brain | 0.5-2 | Abcam | ab53554, ab4674 | Chung et al, Nature 2013; Tomer et al, Nature Protocols 2014; Murray et al, Cell 2015; Costantini et al, Scientific Reports 2015 |
1:400 | Mouse brain | 1 | Aves | GFAP | Yang et al, Cell 2014 | |
1:100-1:500 | Mouse brain | 1 | Cell Signaling Technology | 3670 | Murray et al, Cell 2015; Lee et al, Scientific Reports 2016 | |
1:800 | Mouse brain | 1 | Abcam | ab7260 | Lee et al, Scientific Reports 2016 | |
1:500-1:2000 | Mouse brain | 0.25-1 | Dako | Z0334 | Lee et al, Scientific Reports 2016; Perez et al, J. Clinical Investigation 2017 | |
1:500 | Mouse brain | 1 | Invitrogen | 130330 | Lee et al, Scientific Reports 2016 | |
1:50-1:100 | Mouse brain | 1-2 | Sigma | Bastrup and Larsen, J. Neuro. Methods 2017 | ||
Glucagon | 1:100 | Mouse pancreas; human pancreas | 1+ | Takara | M182 | Hsueh et al, Scientific Reports 2017 |
Glucagon-like peptide 1 (GLP-1) | 1:1500 | Rat brain | 2 | Peninsula Laboratories | T-4363 | Zheng and Rinaman, Brain Struct. Funct. 2016 |
Glucose transporter-1 (GLUT1) | 1:800 | Mouse brain | 1 | Abcam | ab15309 | Lee et al, Scientific Reports 2016 |
1:100-1:300 | Mouse brain; human brain | 0.25-1 | Thermo Fisher | PA1-21041 | Lee et al, Scientific Reports 2016; Phillips et al, Scientific Reports 2016 | |
Glutamic acid decarboxylase-67 (GAD67) | 1:100 | Mouse brain | 1 | Abcam | ab26116 | Murray et al, Cell 2015 |
1:500 | Mouse brain | 1 | Millipore | MAB5406 | Lee et al, Scientific Reports 2016 | |
Green fluorescent protein (GFP) | 1:50-1:100 | Mouse brain; mouse pancreas; human pancreas | 0.3-1+ | Life Technologies | A21311, A31851, A21312, A31852, G10362, A11122 | Chung et al, Nature 2013; Tomer et al, Nature Protocolos 2014; Adhikari et al, Nature 2015; Murray et al, Cell 2015; Li et al, Scientific Reports 2015; Uribe-Marino et al, Biological Psychiatry 2016; Hsueh et al, Scientific Reports 2017 |
1:200-1:600 | Mouse brain; Xenopus brain; Zebrafish | 0.5+ | Aves | GFP-1020, GFP-1010 | Unal et al, J. Neurosci. 2015; Fini et al, Scientific Reports 2017; Mayrohofer et al, Disease Models and Mech. 2017; Xavier et al, J. Comparative Neurology 2017; Passoni et al, Disease Models and Mech. 2017 | |
1:100 | Mouse lung | 0.3 | Novus | Saboor et al, Eur. Respir. J. 2016 | ||
1:500-1:1000 | Mouse brain | 1-3 | Abcam | ab13970 | Plummer et al, Development 2015; Lee et al, Scientific Reports 2016 | |
1:300 | Mouse brain | 1 | Abcam | ab290 | Lee et al, Scientific Reports 2016 | |
1:200 | Mouse brain | 1 | Millipore | AB3080 | Yu et al, Scientific Reports 2017 | |
1:100 | Mouse spleen | Rockland Immunochemicals | 600-101-215 | Murray et al, PLoS One 2017 | ||
Growth associated protein-43 (GAP43) | 1:500 | Mouse brain | 1 | Abcam | ab16053 | Lee et al, Scientific Reports 2016 |
Heme oxygenase-1 (HO-1) | 1:250 | Mouse brain | 1 | Abcam | ab13248 | Lee et al, Scientific Reports 2016 |
Histone H3 | 1:100 | Mouse brain | 1 | Cell Signaling Technology | 4499, 12230 | Murray et al, Cell 2015 |
Abcam | ab1791 | Murray et al, Cell 2015; Kim et al, PNAS 2015 | ||||
HIV-1 p24 | 1:200 | Mouse colon; mouse reproductive tract | Dako |
Kieffer et al, eLife 2017 | ||
HuC/D | 1:50 | Mouse intestine | Molecular Probes | A-21271 | Neckel et al, Scientific Reports 2016 | |
Human CD3 | 1:200 | Mouse colon; mouse reproductive tract | Dako | Kieffer et al, eLife 2017 | ||
Human CD4 | 1:50 | Mouse colon; mouse reproductive tract | Dako | Kieffer et al, eLife 2017 | ||
Inositol 1,4,5-triphosphate 3-kinase A (IP3KA) | 1:500 | Mouse brain | 1 | Santa Cruz Biotechnology | sc-11206 | Lee et al, Scientific Reports 2016 |
Insulin | 1:100-1:500 | Human pancreas; mouse pancreas | 2 | Dako | A0564 | Treweek et al, Nature Protocols 2015; Hsueh et al, Scientific Reports 2017 |
Integrin b4 | 1:200 | Mouse kidney; mouse heart; mouse lung | 1 | Santa Cruz Biotechnology | sc-9090 | Yang et al, Cell 2014 |
Integrin b5 | 1:200 | Mouse kidney; mouse heart; mouse lung | 1 | Santa Cruz Biotechnology | sc-14010 | Yang et al, Cell 2014 |
Ionized calcium-binding adaptor molecule-1 (Iba1) | 1:200 | Mouse brain | 1 | Biocare Medical | CP 290A | Yang et al, Cell 2014 |
1:50-1:500 | Mouse brain; human brain | 1 | Wako | 019-19741 | Murray et al, Cell 2015; Liu et al, Neuropath. Appl. Neurobiol. 2016; Lee et al, Scientific Reports 2016 | |
Keratin-5 | 1:100 | Mouse mammary gland | 1 | BioLegend | 905501 | Lloyd-Lewis et al, Breast Cancer Res. 2016 |
Ki67 | 1:100 | Mouse brain | 1 | Abcam | ab16667 | Murray et al, Cell 2015 |
Laminin | 1:100-1:500 | Mouse brain; mouse liver; mouse skeletal muscle | 1 | Sigma | L9393 | Lee et al, Scientific Reports 2016; Verma et al, Skeletal Muscle Regen. in the Mouse 2016 |
1:100 | Mouse skeletal muscle | Sigma | L0663 | Verma et al, Skeletal Muscle Regen. in the Mouse 2016 | ||
Mouse pancreas; human pancreas | 1+ | Millipore | ab2034 | Hsueh et al, Scientific Reports 2017 | ||
Lectin | 1:50 | Mouse skeletal muscle | Vector Labs | DL-1174, DL-1177 | Verma et al, Skeletal Muscle Regen. in the Mouse 2016 |
Multi-round phenotyping
The stable framework of the clarified tissue, whereby the innate biomolecules are chemically attached to the hydrogel network, enables multiple rounds of molecular phenotyping without damage to the preserved structure. In fact, the clearing solution containing SDS micelles that was originally used for tissue lipid clearing can be used following the first round of immunostaining to denature antibodies, disrupt binding, and wash the first round of antibodies and molecular labels out of the hydrogel-embedded tissue.
Procedure
- Remove sample from imaging chamber, place in 50 mL PBST and incubate at room temperature or 37°C overnight with shaking
- Place sample in 50 mL of clearing solution and incubate at 60°C overnight with shaking to wash out first round of antibodies
- Place sample in 50 mL PBST and incubate at room temperature or 37°C overnight with shaking to wash out clearing solution
- Complete next round of immunostaining and imaging